EP2279747A1 - Vésicules bactériennes immunogènes dotées de protéines de membrane externe - Google Patents

Vésicules bactériennes immunogènes dotées de protéines de membrane externe Download PDF

Info

Publication number
EP2279747A1
EP2279747A1 EP10183544A EP10183544A EP2279747A1 EP 2279747 A1 EP2279747 A1 EP 2279747A1 EP 10183544 A EP10183544 A EP 10183544A EP 10183544 A EP10183544 A EP 10183544A EP 2279747 A1 EP2279747 A1 EP 2279747A1
Authority
EP
European Patent Office
Prior art keywords
vesicles
bacterium
proteins
protein
knockout
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP10183544A
Other languages
German (de)
English (en)
Other versions
EP2279747B1 (fr
Inventor
Jeannette Adu-Bobie
Mariagrazia Pizza
Nathalie Norais
Germano Ferrari
Guido Grandi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GSK Vaccines SRL
Original Assignee
Novartis Vaccines and Diagnostics SRL
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novartis Vaccines and Diagnostics SRL filed Critical Novartis Vaccines and Diagnostics SRL
Priority to PL10183544T priority Critical patent/PL2279747T3/pl
Publication of EP2279747A1 publication Critical patent/EP2279747A1/fr
Application granted granted Critical
Publication of EP2279747B1 publication Critical patent/EP2279747B1/fr
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/095—Neisseria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0258—Escherichia
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention is in the field of vesicle preparation for immunisation purposes.
  • OMVs outer membrane vesicles
  • the 'RIVM' vaccine is based on OMVs containing six different PorA subtypes. It has been shown to be immunogenic in children in phase II clinical trials [2].
  • Reference 3 discloses a vaccine against different pathogenic serotypes of serogroup B meningococcus based on OMVs which retain a protein complex of 65-kDa.
  • Reference 4 discloses a vaccine comprising OMVs from genetically-engineered meningococcal strains, with the OMVs comprising: at least one Class 1 outer-membrane protein (OMP) but not comprising a Class 2/3 OMP.
  • Reference 5 discloses OMVs comprising OMPs which have mutations in their surface loops and OMVs comprising derivatives of meningococcal lipopolysaccharide (LPS).
  • OMP outer-membrane protein
  • vesicles As well as serogroup B N.meningitidis, vesicles have been prepared for other bacteria.
  • Reference 6 discloses a process for preparing OMV-based vaccines for serogroup A meningococcus.
  • References 7 and 8 disclose vesicles from N.gonorrhoeae.
  • Reference 9 discloses vesicle preparations from N.lactamica.
  • Vesicles have also been prepared from Moraxella catarrhalis [10,11], Shigella flexneri [12,13], Pseudomonas aeruginosa [12,13], Porphyromonas gingivalis [14], Treponema pallidum [15], Haemophilus influenzae [16 & 21] and Helicobacter pylori [17].
  • OMVs The failure of OMVs to elicit cross-protection against non-homologous strains is not well understood, particularly as most N.meningitidis isolates share a small number of conserved protective surface antigens that, if present in OMVs, would be expected to provide broad protective coverage.
  • One possible explanation for the failure is the existence of variable immune-dominant surface antigens that prevent the conserved antigens from exerting their protective action, and the presence of immune-dominant hyper-variable proteins such as PorA has been extensively documented and demonstrated.
  • Other possible explanations are that the methods for OMV preparation result in contamination with cytoplasmic and/or inner membrane proteins that dilute the protective outer membrane proteins, or that antigens are lost by the detergent extraction.
  • Reference 18 discloses compositions comprising OMVs supplemented with transferrin binding proteins (e.g . TbpA and TbpB) and/or Cu,Zn-superoxide dismutase.
  • Reference 19 discloses compositions comprising OMVs supplemented by various proteins.
  • Reference 20 discloses preparations of membrane vesicles obtained from N.meningitidis with a modified fur gene.
  • Reference 21 teaches that nspA expression should be up-regulated with concomitant porA and cps knockout. Further knockout mutants of N.meningitidis for OMV production are disclosed in references 21 to 23.
  • reference 24 focuses on changing the methods for OMV preparation, and teaches that antigens such as NspA can be retained during vesicle extraction by avoiding the use of detergents such as deoxycholate.
  • the invention is based on the surprising discovery that disruption of the pathways involved in degradation of peptidoglycan (the murein layer) gives bacteria that release vesicles into their culture medium, and that these vesicles are rich in immunogenic outer membrane proteins and can elicit broad-ranging bactericidal immune responses.
  • the vesicles are different from the OMVs that can be prepared by disrupting whole bacteria ( e.g . by sonication and sarkosyl extraction [25]), and can be prepared without even disrupting bacterial cells e.g . simply by separating the vesicles from the bacteria by a process such as centrifugation.
  • the inventors have found that knockout of the meningococcal mltA homolog (also referred to as 'GNA33' or 'NMB0033' [26]) leads to the spontaneous release of vesicles that are rich in immunogenic outer membrane proteins and that can elicit broadly cross-protective antibody responses with higher bactericidal titres than OMVs prepared by normal production processes.
  • This enhanced efficacy is surprising for two reasons: first, the NMB0033 protein has previously been reported to be highly effective in raising bactericidal antibodies (e.g . see table 1 of ref. 27) and to be a strong vaccine candidate ( e.g . see table 2 of ref.
  • the invention provides a bacterium having a knockout mutation of its mltA gene.
  • the bacterium preferably also has a knockout mutation of at least one further gene e.g. the porA and/or porB and or lpxA genes.
  • the invention also provides a bacterium, wherein: (i) the bacterium has a cell wall that includes peptidoglycan; and (ii) the bacterium does not express a protein having the lytic transglycosylase activity of MItA protein.
  • the bacterium is preferably a mutant bacterium i. e. the bacterium is a mutant strain of a wild-type species that expresses MItA protein.
  • the bacterium preferably also does not express at least one further protein e.g . the PorA and/or PorB and/or LpxA proteins.
  • the invention also provides a composition comprising vesicles that, during culture of bacteria of the invention, are released into the culture medium.
  • This composition preferably does not comprise any living and/or whole bacteria.
  • This composition can be used for vaccine preparation.
  • the invention also provides a composition comprising vesicles, wherein the vesicles are present in the filtrate obtainable after filtration through a 0.22 ⁇ m filter of a culture medium in which a bacterium of the invention has been grown.
  • This composition can be used for vaccine preparation.
  • the invention also provides a process for preparing bacterial vesicles, comprising the steps of: (i) culturing a MltA ⁇ bacterium in a culture medium such that the bacterium releases vesicles into said medium; and (ii) collecting the vesicles from said medium.
  • the MltA ⁇ bacterium is preferably a ⁇ MltA knockout mutant.
  • the vesicles can be collected by size separation (e.g . filtration, using a filter which allows the vesicles to pass through but which does not allow intact bacteria to pass through), which can conveniently be performed after centrifugation to preferentially pellet cells relative to the smaller vesicles ( e.g . low speed centrifugation).
  • Peptidoglycan (also known as murein, mucopeptide or glycosaminopeptide) is a heteropolymer found in the cell wall of most bacteria. Peptidoglycan is the component that is primarily responsible for the mechanical strength of the bacterial cell wall and for maintaining cellular shape. In Gram-positive bacteria it is the major component of the cell wall. In Gram-negative bacteria it occurs as a layer between the cytoplasmic and outer membranes, and is covalently linked to the outer membrane via the Braun lipoprotein.
  • Peptidoglycan consists mainly of linear heteropolysaccharide backbone chains that are cross-linked by 'stem' peptides to form a lattice structure. It is a polymer so large that it can be thought of as a single immense covalently linked molecule.
  • the saccharide backbone is formed from alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues.
  • GlcNAc N-acetylglucosamine
  • MurNAc N-acetylmuramic acid
  • a MurNAc residue may be linked to a stem tetrapeptide.
  • Cross-links between backbone chains are usually formed directly between D-alanine in one stem peptide and a meso -DAP of another.
  • the E.coli structure is typical for Gram-negative bacteria, but there is more variation within Gram-positive bacteria e.g. in S.aureus 30-50% of the muramic acid residues are not acetylated, the stem peptide often has L-lysine in place of meso -DAP and isoglutamine in place of D-glutamate, and cross-links can occur between stem peptides.
  • the initial step in E.coli peptidoglycan biosynthesis is the formation of the UDP derivative of GlcNAc, which occurs in the cytoplasm.
  • Some UDP-GlcNAc is converted to UDP-MurNAc in a reaction of UDP-GlcNAc and phosphoenolpyruvate (PEP), catalysed by PEP:UDP-GlcNAc enolpyruvyl transferase.
  • PEP phosphoenolpyruvate
  • amino acids are added sequentially to UDP-MurNAc to form a UDP-MurNAc-pentapeptide known as the 'Park nucleotide' that includes a terminal D-alanyl-D-alanine.
  • the Park nucleotide is then transferred to bactoprenol monophosphate in the cytoplasmic membrane, where UDP-GlcNAC is also added to make a bactoprenol-disaccharide-pentapeptide subunit.
  • the disaccharide-pentapeptide subunit is then transferred into the periplasmic region, with bactoprenol-pyrophosphate remaining in the membrane. Within the periplasm the transferred subunit is inserted into a growing peptidoglycan.
  • murein hydrolases [30], which as a family includes lytic transglycosylases (mltA, mltB, mltC, mltD, sit 70, emtA), endopeptidases ( pbp4, pbp7, mepA ) and amidases ( amiC ).
  • Muramidases such as lysozyme cleave the same ⁇ -(1-4)-glycosidic linkages between MurNAc and GlcNAc residues; unlike muramidases, however, the transglycosylases cleave the glycosidic bond with concomitant formation of 1,6-anhydromuramoyl residues (AnhMurNAc).
  • the standard peptidoglycan anabolic and catabolic pathways are thus well-characterised, as are the minor variations and modifications that occur between bacteria.
  • the enzymes are well-characterised, and proteins have been readily annotated as being involved in the pathways when new bacterial genomic sequences have been published. The skilled person can thus easily determine the enzymes involved in the peptidoglycan metabolic pathways for any given bacterium, can easily identify the enzymes involved, and can easily identify the genes encoding those enzymes.
  • the invention is based on the knockout of the mltA gene, which encodes a membrane-bound lytic transglycosylase.
  • the MltA family is recognised in INTERPRO (entry 'ipr005300') and PFAM (entry 'MltA' or 'PF03562'), and the PFAM record lists MltA proteins in bacteria as diverse as Rhizobium loti, Bradyrhizobium japonicum, Brucella melitensis, Brucella suis, Rhizobium meliloti, Agrobacterium tumefaciens, Zymomonas mobilis, Caulobacter crescentus, Yersinia pestis, Salmonella typhimurium, Buchnera aphidicola, Photorhabdus luminescens, Escherichia coli, Shigella flexneri, Salmonella typhi, Pseudomonas aeruginosa, Pseudomon
  • Knockout of mltA can result in reduced virulence, abnormal cell separation, abnormal cell morphology, undivided septa, double septa, cell clustering and sharing of outer membranes [25].
  • the knockout mutation has surprisingly been found to give bacteria that can spontaneously produce vesicles that are immunogenic and enriched in outer membrane proteins.
  • the bacterium from which vesicles are prepared may be Gram-positive, but it is preferably Gram-negative.
  • the bacterium may be from genus Moraxella, Shigella, Pseudomonas, Treponema, Porphyromonas or Helicobacter (see above for preferred species) but is preferably from the Neisseria genus.
  • Preferred Neisseria species are N.meningitidis and N.gonorrhoeae.
  • the bacterium may have one or more knockout mutations of other gene(s).
  • the bacterium should have low endotoxin (LOS/LPS) levels, and this can be achieved by knockout of enzymes involved in LPS biosynthesis.
  • LOS/LPS low endotoxin
  • Suitable mutant bacteria are already known e.g . mutant Neisseria [31,32] and mutant Helicobacter [33].
  • the lpxA mutant of meningococcus is preferred. Processes for preparing LPS-depleted outer membranes from Gram-negative bacteria are disclosed in reference 34.
  • the bacterium may express one or more genes that are not endogenous.
  • the invention may use a recombinant strain that expresses new genes relative to the corresponding wild-type strain. Expression of non-endogenous genes in this way can be achieved by various techniques e.g . chromosomal insertion (as used for introducing multiple PorA genes [35]), knockin mutations, expression from extra-chromosomal vectors ( e.g . from plasmids), etc.
  • the bacterium may over-express (relative to the corresponding wild-type strain) immunogens such as NspA, protein 287 [19], protein 741 [36], TbpA [18], TbpB [18], superoxide dismutase [18], etc.
  • the bacterium may also include one or more of the knockout and/or over-expression mutations disclosed in reference 16, 21-24 and/or 37-38.
  • Preferred genes for down-regulation and/or knockout include: (a) Cps, CtrA, CtrB, CtrC, CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc, PilC, PorA, PorB, SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB [16]; (b) CtrA, CtrB, CtrC, CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc, PhoP, PilC, PmrE, PmrF, PorA, SiaA, SiaB, SiaC, SiaD,
  • Vesicles may be prepared from the Escherichia genus, such as from the E.coli species. E.coli strains have traditionally been classified as either commensal or pathogenic, and pathogenic strains are then sub-classified as intestinal or extraintestinal strains. Classification may also be based on the 'K' antigens. The best-studied 'K' antigen is 'K1', which is considered to be the major determinant of virulence among those strains of E.coli that cause neonatal meningitis. Vesicles of the invention can be prepared from any of these E.
  • coli strains are preferably from a pathogenic strain, including an extraintestinal pathogenic ('ExPEC' [39]) strain, a uropathogenic (UPEC) strain or a meningitis/sepsis-associated (MNEC) strains.
  • Genome sequences of pathogenic strains are available in the databases under accession numbers AE005174, BA000007 and NC-004431.
  • the meningococci do not have a homolog of the Tol-Pal system.
  • the invention provides the vesicles that are spontaneously released into culture medium by bacteria of the invention.
  • These vesicles are distinct from the vesicles that can be prepared artificially from the same bacteria, such as the sarkosyl-extracted OMVs prepared in reference 25 from ' ⁇ GNA33' meningococci. They are also distinct from microvesicles (MVs [41]) and 'native OMVs' ('NOMVs' [54]), although vesicles of the invention seem to be more similar to MVs and NOMVs than to sarkosyl-extracted OMVs.
  • the vesicles are also distinct from blebs, which are outer-membrane protrusions that remain attached to bacteria prior to release as MVs [42,43].
  • the vesicles of the invention have a diameter of 50-100nm by electron microscopy, which is smaller than that of artificial meningococcal OMVs (diameter ⁇ 270nm [44]).
  • the diameter is roughly the same as that of artificial OMVs that have been heat-denatured ( ⁇ 105nm [44]), but the vesicles of the invention retain antigenicity whereas heat-denatured artificial OMVs lose their antigenicity.
  • vesicles of the invention (unlike MVs, OMVs and NOMVs) are substantially free from cytoplasmic contamination.
  • Vesicles of the invention preferably contain no more than 20% by weight of LOS/LPS, measured relative to the total protein ( i.e. there should be at least 4x more protein than LOS/LPS, by weight).
  • the maximum LOS/LPS level is preferably even lower than 20% e.g . 15%, 10%, 5% or lower.
  • the vesicle-containing compositions of the invention will generally be substantially free from whole bacteria, whether living or dead.
  • the size of the vesicles of the invention means that they can readily be separated from whole bacteria by filtration through a 0.22 ⁇ m filter e.g . as typically used for filter sterilisation.
  • the invention provides a process for preparing vesicles of the invention, comprising filtering the culture medium from bacteria of the invention through a filter that retards whole bacteria but that lets the vesicles pass through e.g . a 0.22 ⁇ m filter.
  • vesicles will pass through a standard 0.22 ⁇ m filters, these can rapidly become clogged by other material, and so it is preferred to perform sequential steps of filter sterilisation through a series of filters of decreasing pore size, finishing with a standard sterilisation filter (e.g . a 0.22 ⁇ m filter). Examples of preceding filters would be those with pore size of 0.8 ⁇ m, 0.45 ⁇ m, etc.
  • the filtrate can be further treated e.g . by ultracentrifugation.
  • Vesicles of the invention contain lipids and proteins.
  • the protein content of meningococcal vesicles has been analysed, and substantially all of the proteins in the vesicles are classified as outer membrane proteins by bioinformatic analysis.
  • Outer membrane proteins seen in the vesicles include: PilE; IgA-specific serine endopeptidase; PorA; FrpB; P1B; etc.
  • the vesicles of the invention were found to lack proteins such as MinD, FtsA and phosphoenolpyruvate synthase.
  • the vesicles also lack MltA.
  • the vesicles of the invention are advantageous when compared to vesicles prepared by disruption of cultured bacteria because no artificial disruption is required. Simple size-based separation can be used to separate bacteria and vesicles, without any need for chemical treatments, etc. As well as being a simpler process, this avoids the risk of denaturation caused by the detergents etc. that are used during prior art OMV preparative processes.
  • vesicles of the invention may be similar to microvesicles (MVs) and 'native OMVs' ('NOMVs'), which are naturally-occurring membrane vesicles that form spontaneously during bacterial growth and are released into culture medium.
  • MVs can be obtained by culturing Neisseria in broth culture medium, separating whole cells from the broth culture medium (e.g . by filtration or by low-speed centrifugation to pellet only the cells and not the smaller vesicles) and then collecting the MVs that are present in the cell-depleted medium (e.g . by filtration, by differential precipitation or aggregation of MVs, by high-speed centrifugation to pellet the MVs).
  • Strains for use in production of MVs can generally be selected on the basis of the amount of MVs produced in culture. References 46 and 47 describe Neisseria with high MV production.
  • the invention allows the production of immunogenic vesicles from a bacterium of choice.
  • the bacterium will typically have been generated by mutation of a chosen starting strain. Where there are multiple starting strains of interest then the invention provides methods for preparing vesicles from each of the strains, and the different vesicles can be combined. This combination strategy is particularly useful for bacteria where strain-to-strain variation means that a single strain usually does not offer clinically-useful protection e.g . serogroup B meningococcus.
  • the invention provides a composition comprising a mixture of n sets of vesicles of the invention, prepared from n different strains of a bacterium.
  • the value of n can be 1, 2, 3, 4, 5, etc.
  • the different strains can be in the same or different serogroups.
  • the invention also provides a kit comprising vesicles of the invention prepared from n different strains of a bacterium.
  • the vesicles can be kept and stored separately in the kit until they are required to be used together e.g . as an admixture, or for simultaneous separate or sequential use.
  • the invention also provides a process comprising: preparing n sets of vesicles of the invention, one from each of n different strains of a bacterium; and combining the n sets of vesicles.
  • the different sets can be combined into a kit or into an admixture.
  • the invention also provides the use of vesicles from a first strain of a bacterium in the manufacture of a medicament for immunising a patient, wherein the medicament is administered simultaneously separately or sequentially with vesicles from a second strain of the bacterium.
  • the invention also the use of vesicles from a first strain of a bacterium in the manufacture of a medicament for immunising a patient, wherein the patient has been pre-immunised with vesicles from a second strain of the bacterium.
  • Vesicles can be selected from different pathogens.
  • the invention provides a composition comprising a mixture of n sets of vesicles of the invention, prepared from n different species of bacteria.
  • the invention provides a kit comprising vesicles of the invention prepared from n different species of bacteria, and provides a process comprising the step of preparing n sets of vesicles of the invention, one from each of n different species of bacteria.
  • MltA protein expression can be achieved in two main ways: removal or disruption of the endogenous mltA gene (including its control regions) to give a MltA ⁇ strain; or suppression of MltA expression in a MltA + strain. It is preferred to use a MltA ⁇ strain.
  • MltA ⁇ strains can be constructed by conventional knockout techniques. Techniques for gene knockout are well known, and meningococcus knockout mutants of have been reported previously [ e.g . refs. 25 & 48-50].
  • the knockout is preferably achieved by deletion of at least a portion of the coding region (preferably isogenic deletion), but any other suitable technique may be used e.g . deletion or mutation of the promoter, deletion or mutation of the start codon, etc.
  • the bacterium may contain a marker gene in place of the knocked out gene e.g . an antibiotic resistance marker.
  • mRNA encoding the knocked-out protein will be substantially absent and/or its translation will be substantially inhibited ( e.g . to less than 1% of the level of expression that would be seen in the absence of suppression).
  • site-directed mutagenesis of the endogenous mltA gene can be used.
  • Reference 51 discloses mutants of meningococcal MltA in which residues Glu255, Glu323 and Asp362 were mutated and then tested for MltA catalytic activity.
  • An E255G mutant of showed a 50% reduction in activity, and an E323G mutant showed a 70% reduction in activity.
  • Mutagenesis of specific residues within the MltA coding region can therefore be used as a technique to knockout the lytic transglycolase enzymatic activity without knocking out the coding region.
  • the resulting bacterium will be substantially free from MltA enzymatic activity.
  • the invention provides a pharmaceutical composition comprising (a) vesicles of the invention and (b) a pharmaceutically acceptable carrier.
  • the invention also provides a process for preparing such a composition, comprising the step of admixing vesicles of the invention with a pharmaceutically acceptable carrier.
  • Typical 'pharmaceutically acceptable carriers' include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.
  • Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
  • the vaccines may also contain diluents, such as water, saline, glycerol, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, sucrose, and the like, may be present.
  • Sterile pyrogen-free, phosphate-buffered physiologic saline e.g . pH 7.4 is a typical carrier.
  • a thorough discussion of pharmaceutically acceptable excipients is available in reference 52.
  • compositions of the invention will typically be in aqueous form (i. e. solutions or suspensions) rather than in a dried form ( e.g . lyophilised).
  • Aqueous compositions are also suitable for reconstituting other vaccines from a lyophilised form (e.g. a lyophilised Hib conjugate vaccine, a lyophilised meningococcal conjugate vaccine, etc.).
  • the invention provides a kit, which may comprise two vials, or may comprise one ready-filled syringe and one vial, with the aqueous contents of the syringe being used to reactivate the dried contents of the vial prior to injection.
  • compositions of the invention may be presented in vials, or they may be presented in ready-filled syringes.
  • the syringes may be supplied with or without needles.
  • Compositions may be packaged in unit dose form or in multiple dose form.
  • a syringe will generally include a single dose of the composition, whereas a vial may include a single dose or multiple doses. For multiple dose forms, therefore, vials are preferred to pre-filled syringes.
  • Effective dosage volumes can be routinely established, but a typical human dose of the composition has a volume of about 0.5ml e.g . for intramuscular injection.
  • the RIVM OMV-based vaccine was administered in a 0.5ml volume [53] by intramuscular injection to the thigh or upper arm. Similar doses may be used for other delivery routes e.g . an intranasal OMV-based vaccine for atomisation may have a volume of about 100 ⁇ l or about 130 ⁇ l per spray [54], with four sprays administered to give a total dose of about 0.5ml.
  • the pH of the composition is preferably between 6 and 8, and more preferably between 6.5 and 7.5 ( e.g . about 7 or about 7.4).
  • the pH of the RIVM OMV-based vaccine is 7.4 [55], and a pH ⁇ 8 (preferably ⁇ 7.5) is preferred for compositions of the invention.
  • Stable pH may be maintained by the use of a buffer e.g . a Tris buffer, a phosphate buffer, or a histidine buffer.
  • Compositions of the invention will generally include a buffer. If a composition comprises an aluminium hydroxide salt, it is preferred to use a histidine buffer [56] e.g . at between 1-10mM, preferably about 5mM.
  • the RIVM OMV-based vaccine maintains pH by using a 10mM Tris/HCl buffer.
  • the composition may be sterile and/or pyrogen-free.
  • Compositions of the invention may be isotonic with respect to humans.
  • compositions of the invention are immunogenic, and are more preferably vaccine compositions.
  • Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic.
  • Immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen(s), as well as any other components, as needed.
  • 'immunologically effective amount' it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated ( e.g .
  • compositions of the invention will generally be expressed in terms of the amount of protein per dose.
  • a dose of about 0.9 mg protein per ml is typical for OMV-based intranasal vaccines [54].
  • the MeNZBTM OMV-based vaccine contains between 25 and 200 ⁇ g of protein per millilitre e.g . between 45 and 90 ⁇ g/ml, or 50 ⁇ 10 ⁇ g/ml.
  • Compositions of the invention preferably include less than 100 ⁇ g/ml of OMV per strain of bacterium.
  • compositions of the invention may be prepared in various forms.
  • the compositions may be prepared as injectables, either as liquid solutions or suspensions.
  • the composition may be prepared for pulmonary administration e.g . as an inhaler, using a fine powder or a spray.
  • the composition may be prepared as a suppository or pessary.
  • the composition may be prepared for nasal, aural or ocular administration e.g . as spray, drops, gel or powder [ e.g . refs 57 & 58].
  • compositions of the invention may include an antimicrobial, particularly when packaged in multiple dose format.
  • Antimicrobials such as thiomersal and 2-phenoxyethanol are commonly found in vaccines, but it is preferred to use either a mercury-free preservative or no preservative at all.
  • compositions of the invention may comprise detergent e.g . a Tween (polysorbate), such as Tween 80.
  • Detergents are generally present at low levels e.g . ⁇ 0.01%.
  • compositions of the invention may include sodium salts (e.g . sodium chloride) to give tonicity.
  • sodium salts e.g . sodium chloride
  • a concentration of 10+2 mg/ml NaCl is typical.
  • the concentration of sodium chloride is preferably greater than 7.5 mg/ml.
  • compositions of the invention will generally be administered in conjunction with other immunoregulatory agents.
  • compositions will usually include one or more adjuvants, and the invention provides a process for preparing a composition of the invention, comprising the step of admixing vesicles of the invention with an adjuvant e.g . in a pharmaceutically acceptable carrier.
  • adjuvants include, but are not limited to:
  • Mineral containing compositions suitable for use as adjuvants in the invention include mineral salts, such as aluminium salts and calcium salts.
  • the invention includes mineral salts such as hydroxides (e.g . oxyhydroxides), phosphates ( e.g . hydroxyphosphates, orthophosphates), sulphates, etc. [ e.g. see chapters 8 & 9 of ref. 59], or mixtures of different mineral compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous, etc.), and with adsorption being preferred.
  • the mineral containing compositions may also be formulated as a particle of metal salt [60].
  • a typical aluminium phosphate adjuvant is amorphous aluminium hydroxyphosphate with PO 4 /Al molar ratio between 0.84 and 0.92, included at 0.6mg Al 3+ /ml.
  • Adsorption with a low dose of aluminium phosphate may be used e.g . between 50 and 100 ⁇ g Al 3+ per conjugate per dose.
  • an aluminium phosphate it used and it is desired not to adsorb an antigen to the adjuvant, this is favoured by including free phosphate ions in solution ( e.g . by the use of a phosphate buffer).
  • the RIVM vaccine was tested with adsorption to either an aluminium phosphate or an aluminium hydroxide adjuvant, and the aluminium phosphate adjuvant was found to give superior results [55].
  • the MeNZBTM, MenBvacTM abd VA-MENINGOC-BCTM products all include an aluminium hydroxide adjuvant.
  • a typical dose of aluminium adjuvant is about 3.3 mg/ml (expressed as Al 3+ concentration).
  • Oil emulsion compositions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59 [Chapter 10 of ref. 59; see also ref. 61] (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer). Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may also be used.
  • CFA Complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • Saponin formulations may also be used as adjuvants in the invention.
  • Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria officianalis (soap root).
  • Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs. QS21 is marketed as StimulonTM.
  • Saponin compositions have been purified using HPLC and RP-HPLC. Specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C.
  • the saponin is QS21.
  • a method of production of QS21 is disclosed in ref. 62.
  • Saponin formulations may also comprise a sterol, such as cholesterol [63].
  • ISCOMs immunostimulating complexs
  • the ISCOM typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs.
  • the ISCOM includes one or more of QuilA, QHA and QHC. ISCOMs are further described in refs. 63-65.
  • the ISCOMS may be devoid of extra detergent [66].
  • Virosomes and virus-like particles can also be used as adjuvants in the invention.
  • These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genome.
  • the viral proteins may be recombinantly produced or isolated from whole viruses.
  • viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, Q ⁇ -phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein pl).
  • VLPs are discussed further in refs. 69-74.
  • Virosomes are discussed further in, for example, ref. 75
  • Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), Lipid A derivatives, immunostimulatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives thereof.
  • LPS enterobacterial lipopolysaccharide
  • Lipid A derivatives Lipid A derivatives
  • immunostimulatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives thereof.
  • Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL).
  • 3dMPL is a mixture of 3 de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains.
  • a preferred "small particle" form of 3 De-O-acylated monophosphoryl lipid A is disclosed in ref. 76. Such "small particles" of 3dMPL are small enough to be sterile filtered through a 0.22 ⁇ m membrane [76].
  • Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g . RC-529 [77,78].
  • Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174.
  • OM-174 is described for example in refs. 79 & 80.
  • Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine). Double-stranded RNAs and oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory.
  • the CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded.
  • References 81, 82 and 83 disclose possible analog substitutions e.g . replacement of guanosine with 2'-deoxy-7-deazaguanosine.
  • the adjuvant effect of CpG oligonucleotides is further discussed in refs. 84-89.
  • the CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT [90].
  • the CpG sequence may be specific for inducing a Thl immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN.
  • CpG-A and CpG-B ODNs are discussed in refs. 91-93.
  • the CpG is a CpG-A ODN.
  • the CpG oligonucleotide is constructed so that the 5' end is accessible for receptor recognition.
  • two CpG oligonucleotide sequences may be attached at their 3' ends to form "immunomers". See, for example, refs. 90 & 94-96.
  • Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention.
  • the protein is derived from E.coli ( E.coli heat labile enterotoxin "LT"), cholera ("CT"), or pertussis ("PT").
  • LT E.coli heat labile enterotoxin
  • CT cholera
  • PT pertussis
  • the use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in ref. 97 and as parenteral adjuvants in ref. 98.
  • the toxin or toxoid is preferably in the form of a holotoxin, comprising both A and B subunits.
  • the A subunit contains a detoxifying mutation; preferably the B subunit is not mutated.
  • the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LT-G192.
  • LT-K63 LT-K63
  • LT-R72 LT-G192.
  • ADP-ribosylating toxins and detoxified derivaties thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in refs. 99-106.
  • Numerical reference for amino acid substitutions is preferably based on the alignments of the A and B subunits of ADP-ribosylating toxins set forth in ref. 107, specifically incorporated herein by reference in its entirety.
  • Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 [108], etc.) [109], interferons (e.g. interferon- ⁇ ), macrophage colony stimulating factor, and tumor necrosis factor.
  • cytokines such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 [108], etc.) [109], interferons (e.g. interferon- ⁇ ), macrophage colony stimulating factor, and tumor necrosis factor.
  • Bioadhesives and mucoadhesives may also be used as adjuvants in the invention.
  • Suitable bioadhesives include esterified hyaluronic acid microspheres [110] or mucoadhesives such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants in the invention [111].
  • Microparticles may also be used as adjuvants in the invention.
  • Microparticles i.e. a particle of ⁇ 100nm to ⁇ 150 ⁇ m in diameter, more preferably ⁇ 200nm to ⁇ 30 ⁇ m in diameter, and most preferably ⁇ 500nm to ⁇ 10 ⁇ m in diameter
  • materials that are biodegradable and non-toxic e.g . a poly( ⁇ -hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.
  • a negatively-charged surface e.g. with SDS
  • a positively-charged surface e.g. with a cationic detergent, such as CTAB
  • liposome formulations suitable for use as adjuvants are described in refs. 112-114.
  • Adjuvants suitable for use in the invention include polyoxyethylene ethers and polyoxyethylene esters [115]. Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol [116] as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol [117].
  • Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.
  • PCPP Polyphosphazene
  • PCPP formulations are described, for example, in refs. 118 and 119.
  • muramyl peptides suitable for use as adjuvants in the invention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE).
  • thr-MDP N-acetyl-muramyl-L-threonyl-D-isoglutamine
  • nor-MDP N-acetyl-normuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-
  • imidazoquinolone compounds suitable for use adjuvants in the invention include Imiquamod and its homologues (e,g. "Resiquimod 3M"), described further in refs. 120 and 121.
  • the invention may also comprise combinations of aspects of one or more of the adjuvants identified above.
  • the following adjuvant compositions may be used in the invention: (1) a saponin and an oil-in-water emulsion [122]; (2) a saponin (e.g . QS21) + a non-toxic LPS derivative (e.g. 3dMPL) [123]; (3) a saponin ( e.g . QS21) + a non-toxic LPS derivative (e.g . 3dMPL) + a cholesterol; (4) a saponin ( e.g .
  • Ribi TM adjuvant system (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (DetoxTM); and (8) one or more mineral salts (such as an aluminum salt) + a non-toxic derivative of LPS (such as 3dMPL).
  • MPL monophosphorylipid A
  • TDM trehalose dimycolate
  • CWS cell wall skeleton
  • LPS such as 3dMPL
  • aluminium salt adjuvants is particularly preferred, and antigens are generally adsorbed to such salts. It is possible in compositions of the invention to adsorb some antigens to an aluminium hydroxide but to have other antigens in association with an aluminium phosphate. In general, however, it is preferred to use only a single salt e.g . a hydroxide or a phosphate, but not both. Not all vesicles need to be adsorbed i. e. some or all can be free in solution.
  • the invention also provides a method for raising an immune response in a mammal, comprising administering a pharmaceutical composition of the invention to the mammal.
  • the immune response is preferably protective and preferably involves antibodies.
  • the method may raise a booster response in a patient that has already been primed against N.meningitidis. Subcutaneous and intranasal prime/boost regimes for OMVs are disclosed in ref. 126.
  • the mammal is preferably a human.
  • the human is preferably a child (e.g . a toddler or infant) or a teenager; where the vaccine is for therapeutic use, the human is preferably an adult.
  • a vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc .
  • the invention also provides vesicles of the invention for use as a medicament.
  • the medicament is preferably able to raise an immune response in a mammal (i.e. it is an immunogenic composition) and is more preferably a vaccine.
  • the invention also provides the use of vesicles of the invention in the manufacture of a medicament for raising an immune response in a mammal.
  • the invention also the use of vesicles of the invention in the manufacture of a medicament for immunising a patient, wherein the patient has been pre-immunised with at least one of the following: diphtheria toxoid; tetanus toxoid; acellular or cellular pertussis antigens; a conjugated Hib capsular saccharide; hepatitis B virus surface antigen; a conjugated meningococcal capsular saccharide; and/or a conjugated pneumococcal capsular saccharide.
  • compositions of the invention can confer an antibody titre in a patient that is superior to the criterion for seroprotection for an acceptable percentage of human subjects.
  • Antigens with an associated antibody titre above which a host is considered to be seroconverted against the antigen are well known, and such titres are published by organisations such as WHO.
  • Preferably more than 80% of a statistically significant sample of subjects is seroconverted, more preferably more than 90%, still more preferably more than 93% and most preferably 96-100%.
  • compositions of the invention will generally be administered directly to a patient.
  • Direct delivery may be accomplished by parenteral injection (e.g . subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary or other mucosal administration.
  • Intramuscular administration to the thigh or the upper arm is preferred.
  • Injection may be via a needle (e.g . a hypodermic needle), but needle-free injection may alternatively be used.
  • a typical intramuscular dose is 0.5 ml.
  • Dosage treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. A primary dose schedule may be followed by a booster dose schedule. Suitable timing between priming doses ( e.g . between 4-16 weeks), and between priming and boosting, can be routinely determined.
  • the OMV-based RIVM vaccine was tested using a 3- or 4-dose primary schedule, with vaccination at 0, 2 & 8 or 0, 1, 2 & 8 months. MeNZBTM is administered as three doses at six week intervals. These schedules can be used according to the invention.
  • the vesicle preparations given at each dose stage can be the same or different.
  • a second and a third dose may be given over the next two months, and a fourth dose may be given between 11 and 13 months after time zero.
  • the first, second and third doses may comprise vesicles with the same serosubtype as each other, and the fourth dose may comprises vesicles with a different serosubtype from the first three doses.
  • the fourth dose may contain vesicles only with a different serosubtype from the first three doses, or it may contain two types of vesicle, one with a different serosubtype from the first three doses and one with the same subtype.
  • the first, second and third doses are preferably given at intervals of between 6 and 8 weeks.
  • the fourth dose is preferably given about 1 year after time zero.
  • the patient preferably receives the same quantity of vaccine at each of the four doses.
  • the invention may involve administration of vesicles from more than one subtype and/or serosubtype of N.meningitidis [ e.g. ref. 41], either separately or in admixture.
  • the invention may be used to elicit systemic and/or mucosal immunity.
  • compositions of the invention are able to induce serum bactericidal antibody responses after being administered to a subject. These responses are conveniently measured in mice and are a standard indicator of vaccine efficacy [ e.g. see end-note 14 of reference 27].
  • Serum bactericidal activity measures bacterial killing mediated by complement, and can be assayed using human or baby rabbit complement. WHO standards require a vaccine to induce at least a 4-fold rise in SBA in more than 90% of recipients.
  • MeNZBTM elicits a 4-fold rise in SBA 4-6 weeks after administration of the third dose.
  • compositions of the invention may include further non-vesicular antigens.
  • the composition may comprise one or more of the following further antigens:
  • a saccharide or carbohydrate antigen is used, it is preferably conjugated to a carrier in order to enhance immunogenicity. Conjugation of H.influenzae B, meningococcal and pneumococcal saccharide antigens is well known.
  • Toxic protein antigens may be detoxified where necessary (e.g . detoxification of pertussis toxin by chemical and/or genetic means [137]).
  • diphtheria antigen is included in the composition it is preferred also to include tetanus antigen and pertussis antigens. Similarly, where a tetanus antigen is included it is preferred also to include diphtheria and pertussis antigens. Similarly, where a pertussis antigen is included it is preferred also to include diphtheria and tetanus antigens. DTP combinations are thus preferred.
  • Saccharide antigens are preferably in the form of conjugates.
  • Preferred carrier proteins for conjugates are bacterial toxins or toxoids, such as diphtheria toxoid or tetanus toxoid.
  • the CRM197 mutant of diphtheria toxin [158-160] is a particularly preferred carrier for, as is a diphtheria toxoid.
  • suitable carrier proteins include the N.meningitidis outer membrane protein [161], synthetic peptides [162,163], heat shock proteins [164,165], pertussis proteins [166,167], cytokines [168], lymphokines [168], hormones [168], growth factors [168], artificial proteins comprising multiple human CD4 + T cell epitopes from various pathogen-derived antigens [169] such as N19, protein D from H.influenzae [170,171], pneumococcal surface protein PspA [172], pneumolysin [173], iron-uptake proteins [174], toxin A or B from C.difficile [175], etc.
  • vesicles that are not vesicles of the invention e.g . OMVs, MVs, NOMVs, etc. that are prepared by methods other than those of the invention (e.g. prepared by methods involving disruption of bacterial membranes, as disclosed in the prior art).
  • Antigens in the composition will typically be present at a concentration of at least 1 ⁇ g/ml each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen.
  • nucleic acid encoding the antigen may be used.
  • Protein components of the compositions of the invention may thus be replaced by nucleic acid (preferably DNA e.g . in the form of a plasmid) that encodes the protein.
  • composition comprising X may consist exclusively of X or may include something additional e.g . X + Y.
  • references to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids are the same in comparing the two sequences.
  • This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of reference 176.
  • a preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62.
  • the Smith-Waterman homology search algorithm is well known and is disclosed in reference 177.
  • 'GNA33', 'NMB0033' and 'mltA' can be used interchangeably when referring to meningococcus.
  • a meningococcal strain was prepared in which the mltA gene is replaced by allelic exchange with an antibiotic cassette.
  • N.meningitidis strain MC58 was transformed with plasmid pBSUDGNA33ERM.
  • This plasmid contains upstream and downstream flanking regions for allelic exchange, a truncated mltA gene, and the ermC gene (encoding erythromycin resistance).
  • the upstream flanking region (including the start codon) from position -867 to +75 and the downstream flanking region (including the stop codon) from position +1268 to +1744 were amplified from MC58 by using the primers U33FOR, U33REV, D33FOR and D33REV [25]. Fragments were cloned into pBluescriptTM and transformed into E.coli DH5 by using standard techniques.
  • Neisseria strain MC58 was transformed by selecting a few colonies grown overnight on GC agar plates and mixing them with 20 ⁇ l 10 mM Tris-HCl (pH 6.5) containing 1 ⁇ g plasmid DNA. The mixture was spotted onto a chocolate agar plate, incubated for 6 h at 37°C with 5% CO 2 , and then diluted in phosphate buffered-saline (PBS) and spread on GC agar plates containing 7 ⁇ g/ml erythromycin. Allelic exchange with the chromosomal mltA gene was verified by PCR, and lack of MltA expression was confirmed by Western blot analysis.
  • PBS phosphate buffered-saline
  • the ⁇ mltA knockout strain does not have the correct topological organisation of the cellular membrane, has abnormal cell separation, abnormal cell morphology, undivided septa, double septa, cell clustering, sharing of outer membranes and reduced virulence.
  • Reference 25 also reports that the knockout strain releases various membrane proteins into the culture supernatant, including the PorA, PIB, class 4 and class 5 outer membrane proteins.
  • a ⁇ mltA knockout was also made from New Zealand strain 394/98 (lin3; B:4:P1.4), which is the strain from which the MeNZBTM product is produced.
  • the ⁇ mltA strain was grown in GC culture medium in a humidified atmosphere containing 5%CO 2 until OD 600nm 0.5. Bacteria were collected by 10 minutes of centrifugation at 3500 x g. The supernatant (i.e. culture medium) was filtered through a 0.22 ⁇ m pore size filter (Millipore), and the cell-free filtrate was subjected to high-speed centrifugation (200,000 x g, 90 min). This centrifugation resulted in formation of a pellet, with about 8-12 mg protein per litre of culture medium. No such pellet was seen if wild-type MC58 bacteria were treated in the same way, and so the pellet formation is a result of the ⁇ mltA knockout. The pellet was washed twice with PBS (centrifugation 200,000 x g, 30 min) for further analysis.
  • PBS centrifugation 200,000 x g, 30 min
  • Figure 5 shows SDS-PAGE analysis of culture media after growth of wild-type or ⁇ GNA33 bacteria, and shows the different protein release characteristics.
  • the ⁇ mltA-derived vesicles were compared to meningococcal vesicles prepared by the 'normal' detergent extraction method.
  • Meningococcal strains MC58, NZ394/98 and NZ98/254, and their respective isogenic ⁇ mltA mutants were grown in 20 ml or 200 ml GC culture medium in humidified atmosphere containing 5% CO 2 until OD 620nm 0.5. Bacteria were collected by 10-minute centrifugation at 3500g. Vesicles ('DOMVs') were prepared from the wild-type bacteria by detergent extraction as described in reference 178.
  • Vesicles of the invention were prepared from knockout strains by filtration through a 0.22 ⁇ m pore size filter, followed by high-speed centrifugation (200,000g, 90 min) of the filtrates, washing of the vesicle-containing pellets (centrifugation 200,000g, 30 min) twice with phosphate buffer saline, (PBS), and then re-suspension with PBS.
  • PBS phosphate buffer saline
  • Both the mOMVs and the DOMVs were analysed by denaturing mono-dimensional electrophoresis. Briefly, 20 ⁇ g of vesicle proteins were resolved by SDS-PAGE and visualised by Coomassie Blue staining of 12.5% gels. Denaturing (2% SDS) and semi-denaturing (0.2% SDS, no dithiothreitol, no heating) conditions were used mono-dimensional electrophoresis. The amount of protein (20 ⁇ g) was determined by DC protein assay (Bio-Rad), using bovine serum albumin as a standard protein.
  • the vesicles were denatured for 3 minutes at 95°C in SDS-PAGE sample buffer containing 2% SDS. 20 ⁇ g of protein were then loaded onto 12,5% acrylamide gels, which were stained with Coomassie Blue R-250. 2-dimensional electrophoresis was also performed on 200 ⁇ g of protein brought to a final volume of 125 ⁇ l with re-swelling buffer containing 7M urea, 2M thiourea, 2% (w/v) (3-((3-cholamidopropyl)dimethylammonio)-1-propane-sulfonate), 65 mM dithiothreitol, 2% (w/v) amidosulfobetain-14, 2 mM tributylphosphine, 20mM Tris, and 2% (v/v) carrier ampholyte.
  • Proteins were adsorbed overnight onto Immobiline DryStrips (7 cm; pH-gradient 3-10 non linear). Proteins were then 2D-separated.
  • the first dimension was run using a IPGphor Isoelectric Focusing Unit, applying sequentially 150 V for 35 min., 500 V for 35 min., 1,000 V for 30 min, 2,600 V for 10 min., 3,500 V for 15 min., 4,200 V for 15 min., and finally 5,000 V to reach 12kVh.
  • the strips were equilibrated and proteins were separated on linear 9-16.5% polyacrylamide gels (1.5-mm thick, 4 x 7 cm ). Gels were again stained with Coomassie Brilliant Blue G-250. 266 protein spots could be seen after Colloidal Coomassie Blue staining ( Figure 4 ).
  • the 1D and 2D gels were then subjected to in-gel protein digestion and sample preparation for mass spectrometry analysis.
  • Protein spots were excised from the gels, washed with 100 mM ammonium bicarbonate/acetonitrile 50/50 (V/V), and dried using a SpeedVac centrifuge. Dried spots were digested 2 hours at 37°C in 12 ⁇ l of 0.012 ⁇ g/ ⁇ l sequencing grade trypsin (Promega) in 50 mM ammonium bicarbonate, 5 mM. After digestion, 5 ⁇ l of 0.1 % trifluoacetic acid was added, and the peptides were desalted and concentrated with ZIP-TIPs (C18, Millipore).
  • Spectra were externally calibrated using a combination of four standard peptides, angiotensin II (1,046.54 Da), substance P (1,347.74 Da), Bombensin (1,619.82 Da) and ACTH18-39 Clip human (2,465.20 Da), spotted onto adjacent position to the samples. Protein identification was carried out by both automatic and manual comparison of experimentally-generated monoisotopic values of peptides in the mass range of 700-3000 Da with computer-generated fingerprints using the Mascot software.
  • Results from the MC58 ⁇ mltA mutant are shown in Figure 11 .
  • 25 unique proteins were identified, 24 (96%) of which were predicted to be outer-membrane proteins by the PSORT algorithm (Table 1 of W02006/046143 ).
  • 44/51 identified proteins have been assigned to the outer membrane compartment by the genome annotation [Error! Bookmark not defined.] .
  • the 7 remaining proteins were analysed for possible errors in the original annotation.
  • the combined 1D and 2D electrophoresis experiments identified a total of 65 proteins in the MC58 ⁇ mltA mutant-derived vesicles. Of these, 6 proteins were identified in both 1D and 2D gels, whereas 14 and 45 were specific for the 1D and 2D gels, respectively (Table 1 of WO2006/046143 ). Moreover, 61 out of the 65 identified proteins were predicted as membrane-associated proteins by current algorithms, indicating that the ⁇ mltA vesicles (mOMVs) are mostly, and possible exclusively, constituted by membrane proteins.
  • mOMVs ⁇ mltA vesicles
  • Table 2 of W02006/046143 shows 66 proteins that were identified in one or both of the gels, together with the predicted location of the proteins. Again, most of the proteins were predicted as membrane-associated.
  • the 47 proteins common to Tables 1 and 2 of W02006/046143 are shown in Table 3 of WO2006/046143 .
  • Results from the NZ98/254 ⁇ mltA mutant are shown in Figure 12 .
  • 66 proteins were identified from these two gels, 57 of which were assigned to the outer membrane compartment. Again, therefore, the mOMVs are highly enriched in outer membrane proteins. 46 of the 57 proteins had also been identified in the MC58-derived mOMVs.
  • Figure 13 shows the results from NZ98/254 DOMVs.
  • Proteomic analysis revealed 138 proteins, only 44 of which were assigned to the outer membrane compartment. The remaining 94 proteins belonged to the cytoplasmic and inner membrane compartments. Of these 44 membrane proteins, 32 were also found in the 57 outer membrane proteins found in the mOMVs from the isogenic strain.
  • DOMVs are largely constituted by outer membrane proteins, therefore, about 70% of DOMV proteins are either cytoplasmic or inner membrane proteins. DOMVs differ from mOMVs not only for the proportion of cytoplasmic proteins but also for the different profile of their outer membrane proteins. Of the 44 outer membrane proteins seen in DOMVs, only 32 were also seen in mOMVs.
  • a total cell extract of bacteria was prepared as follows: Bacterial cells were washed with PBS, and the bacterial pellet was resuspended in 8 ml of 50 mM Tris-HCl pH 7.3 containing protease inhibitor cocktail (Roche Diagnostic). 2 mM EDTA and 2000 units of benzonase (Merck) were added, cells were disrupted at 4°C with Basic Z 0.75V Model Cell Disrupter equipped with an "one shot head" (Constant System Ltd) by 2 cycles, and the unbroken cells were removed by centrifugation10 min at 8 000 x g at 4°C. This extract was analysed by SDS-PAGE, for comparison with a protein extract of the vesicles produced by ⁇ GNA33 bacteria.
  • porins PorA and PorB are seen in the wild-type bacterial outer membrane (lanes 2 & 4) and also in the ⁇ GNA33 knockout mutant's vesicles (lanes 3 & 5). Moreover, these proteins are retained as stable trimers in the vesicles that do not dissociate into monomers in SDS-PAGE sample buffer with a low concentration of SDS (0.2%) under seminative conditions (no heating before electrophoresis; lanes 2 & 3), but that do denature at 95°C (lanes 4 & 5).
  • LPS levels in detergent-extracted OMVs are typically 5-8% by weight, relative to protein [180].
  • endotoxin content of the vesicles was about twice as high as found in detergent-extracted OMVs.
  • ⁇ mltA-derived vesicles are highly enriched in outer membrane proteins, their ability to elicit bactericidal antibodies capable of killing a broad panel of MenB clinical isolates was investigated.
  • the strain chosen for the testing was 394/98. This strain was chosen because it is the strain from which the MeNZBTM OMV-based vaccine is prepared, thereby aiding a direct comparison of ⁇ mltA vesicles of the invention with OMVs prepared from the wild-type strain by typical prior art methods.
  • mice 10 ⁇ g of each type of vesicle was adsorbed to an aluminium hydroxide adjuvant (3mg/ml) and injected into mice 5-week old CD1 female mice (5-10 mice per group). The vesicles were given intraperitoneally on days 0 and 21. Blood samples for analysis were taken on day 34, and were tested for SBA against 15 different serogroup B strains corresponding to 11 different sub-types, including the four major hypervirulent lineages, using pooled baby rabbit serum as the complement source. Serum bactericidal titers were defined as the serum dilution resulting in 50% decrease in colony forming units (CFU) per ml after 60 minutes incubation of bacteria with reaction mixture, compared to control CFU per ml at time 0.
  • CFU colony forming units
  • bacteria incubated with the negative control antibody in the presence of complement showed a 150 to 200% increase in CFU/ml during the 60 min incubation.
  • Titers were as follows, expressed as the reciprocal of the serum dilution yielding ⁇ 50% bacterial killing: BCA titer Serogroup:Type:Subtype mOMVs DOMVs B:4:P1.4 >8192 >32768 B:15:P1.7,4 >65536 32768 B:4,7:P1.7,4 >32768 >32768 B:14:P1.4 >32768 >65536 B:4:P1.7,4 >32768 8192 B:4,:P1.4 >8192 >8192 B:14:P1.13 16384 512 B:4,7:P1.7,13 >8192 128 B:4:P1.15 >8192 128 B:21:P1.9 >8192 ⁇ 16 B:2b:P1.10 1024 ⁇ 16 B:4,7:P1.19,15 1024 ⁇ 16 B:2
  • mOMVs are better than DOMVs for eliciting complement-dependent antibody killing when tested over a panel of 15 different serogroup B strains.
  • the anti-mOMV mouse sera showed high bactericidal activities against the homologous strain and against 14 additional strains, including 10 different PorA subtypes.
  • mouse sera raised against DOMVs show high bactericidal titers only against six MenB strains, belonging to two PorA subtypes.
  • ⁇ mltA -derived vesicles do contain conserved, protective antigens, they were run on an SDS-PAGE, transferred onto a PDF filter and immunoblotted using specific anti-sera against six proteins antigens previously shown to be protective and highly conserved, including '287', '953', '741' (GNA1870) and 'NadA'.
  • the vesicles were separated onto 10% acrylamide SDS-PAGE gels employing a Mini-Protean II electrophoresis apparatus (Bio-Rad). After protein separation, gels were equilibrated with 48 mM Tris-HCl, 39 mM glycine, pH 9.0, 20% (v/v) methanol and transferred to a nitrocellulose membrane (Bio-Rad) using a Trans-BlotTM semi-dry electrophoretic transfer cell. The nitrocellulose membranes were blocked with 10% (w/v) skimmed milk in PBS containing 0.2% (w/v) sodium azide.
  • the ⁇ mltA -derived vesicles are predominantly constituted by outer membrane proteins, whereas DOMVs are heavily contaminated by cytoplasmic proteins.
  • sera raised against ⁇ mltA -derived vesicles showed a higher and wider strain coverage than DOMVs.
  • a knockout strain of ExPEC CFT073 was prepared by isogenic deletion of the tolR gene, replacing it with a kanamycin resistance marker.
  • the knockout strain was grown to OD 600nm 0.4, and the culture was then centrifuged. The supernatant was filtered through a 0.22 ⁇ m filter and the filtrate was precipitated using TCA. The pellet was then resuspended in Tris buffer.
  • ExPEC strains were prepared from strains DH5a, 536 and IHE3034. Vesicles were prepared as before, and SDS-PAGE analysis of TCA precipitates is shown in Figure 10 .
  • the knockout mutant produces high amounts of vesicles, and these vesicles were subjected to proteomic analyses, including 1D and 2D SDS-PAGE and tryptic digestion of surface-exposed proteins in the vesicles followed by sequence analysis of released peptides.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
EP10183544.5A 2004-10-29 2005-10-28 Vésicules bactériennes immunogènes dotées de protéines de membrane externe Active EP2279747B1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PL10183544T PL2279747T3 (pl) 2004-10-29 2005-10-28 Immunogenne pęcherzyki bakteryjne z białkami błony zewnętrznej

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB0424092.5A GB0424092D0 (en) 2004-10-29 2004-10-29 Immunogenic bacterial vesicles with outer membrane proteins
EP05801788A EP1804834B1 (fr) 2004-10-29 2005-10-28 Vesicules bacteriennes immunogenes comprenant des proteines de la membrane externe

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
EP05801788.0 Division 2005-10-28
EP05801788A Division EP1804834B1 (fr) 2004-10-29 2005-10-28 Vesicules bacteriennes immunogenes comprenant des proteines de la membrane externe

Publications (2)

Publication Number Publication Date
EP2279747A1 true EP2279747A1 (fr) 2011-02-02
EP2279747B1 EP2279747B1 (fr) 2014-06-25

Family

ID=33515801

Family Applications (2)

Application Number Title Priority Date Filing Date
EP10183544.5A Active EP2279747B1 (fr) 2004-10-29 2005-10-28 Vésicules bactériennes immunogènes dotées de protéines de membrane externe
EP05801788A Active EP1804834B1 (fr) 2004-10-29 2005-10-28 Vesicules bacteriennes immunogenes comprenant des proteines de la membrane externe

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP05801788A Active EP1804834B1 (fr) 2004-10-29 2005-10-28 Vesicules bacteriennes immunogenes comprenant des proteines de la membrane externe

Country Status (15)

Country Link
US (3) US9206399B2 (fr)
EP (2) EP2279747B1 (fr)
JP (4) JP5362221B2 (fr)
CN (1) CN101048175B (fr)
AT (1) ATE510559T1 (fr)
AU (1) AU2005298332B2 (fr)
BR (1) BRPI0517514A8 (fr)
CA (1) CA2584778C (fr)
CY (1) CY1112105T1 (fr)
ES (1) ES2507499T3 (fr)
GB (1) GB0424092D0 (fr)
NZ (1) NZ553989A (fr)
PL (1) PL2279747T3 (fr)
PT (1) PT2279747E (fr)
WO (1) WO2006046143A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3263695A1 (fr) 2016-06-29 2018-01-03 GlaxoSmithKline Biologicals SA Compositions immunogènes

Families Citing this family (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK2270173T3 (en) 1999-05-19 2016-03-07 Glaxosmithkline Biolog Sa Neisserial combination compositions
GB9928196D0 (en) 1999-11-29 2000-01-26 Chiron Spa Combinations of B, C and other antigens
MX339524B (es) 2001-10-11 2016-05-30 Wyeth Corp Composiciones inmunogenicas novedosas para la prevencion y tratamiento de enfermedad meningococica.
GB0316560D0 (en) 2003-07-15 2003-08-20 Chiron Srl Vesicle filtration
GB0424092D0 (en) 2004-10-29 2004-12-01 Chiron Srl Immunogenic bacterial vesicles with outer membrane proteins
ES2385045T3 (es) 2005-02-18 2012-07-17 Novartis Vaccines And Diagnostics, Inc. Inmunógenos de Escherichia coli uropatogénica
EP1872791A1 (fr) * 2006-06-30 2008-01-02 Institut Pasteur Utilisation de polysaccharides bactériens pour empêcher la formation d'un film biologique
EP2586790A3 (fr) 2006-08-16 2013-08-14 Novartis AG Immunogènes d'Escherischia coli pathogènes des voies urinaires
AR064642A1 (es) 2006-12-22 2009-04-15 Wyeth Corp Polinucleotido vector que lo comprende celula recombinante que comprende el vector polipeptido , anticuerpo , composicion que comprende el polinucleotido , vector , celula recombinante polipeptido o anticuerpo , uso de la composicion y metodo para preparar la composicion misma y preparar una composi
GB0816284D0 (en) * 2008-09-05 2008-10-15 Nat Biolog Standards Board Vaccine
CN102300585A (zh) 2008-12-17 2011-12-28 诺华有限公司 包含血红蛋白受体的脑膜炎球菌疫苗
GB0917003D0 (en) 2009-09-28 2009-11-11 Novartis Vaccines Inst For Global Health Srl Purification of bacterial vesicles
WO2011039631A2 (fr) 2009-09-30 2011-04-07 Novartis Ag Expression de polypeptides fhbp méningococciques
WO2011043538A2 (fr) * 2009-10-08 2011-04-14 주식회사이언메딕스 Composition comprenant des vésicules de membranes extracellulaires issues de l'air intérieur, et utilisation de celle-ci
GB201009861D0 (en) 2010-06-11 2010-07-21 Novartis Ag OMV vaccines
SI3246044T2 (sl) 2010-08-23 2024-06-28 Wyeth Llc Stabilne formulacije antigenov neisseria meningitidis RLP2086
GB201014967D0 (en) * 2010-09-09 2010-10-20 Univ Southampton Composition
CA2810971C (fr) * 2010-09-10 2020-11-03 Novartis Ag Developpements apportes a des vesicules membranaires externes meningococciques
ES2585328T5 (es) 2010-09-10 2022-12-14 Wyeth Llc Variantes no lipidadas de antígenos ORF2086 de Neisseria meningitidis
EP2707009A1 (fr) 2011-05-12 2014-03-19 Novartis AG Antipyrétiques pour améliorer la tolérabilité de vaccins à base de vésicules
US20140336366A1 (en) * 2011-09-06 2014-11-13 Glycovaxyn Ag Bioconjugate vaccines made in prokaryotic cells
ES2654613T3 (es) * 2012-02-02 2018-02-14 Glaxosmithkline Biologicals Sa Promotores para una expresión aumentada de proteínas en meningococos
EP2823312B1 (fr) 2012-03-08 2019-08-07 GlaxoSmithKline Biologicals SA In vitro essai d'activite des vaccins a base de proteines méningococciques
SA115360586B1 (ar) 2012-03-09 2017-04-12 فايزر انك تركيبات لعلاج الالتهاب السحائي البكتيري وطرق لتحضيرها
RU2665841C2 (ru) 2012-03-09 2018-09-04 Пфайзер Инк. Композиции neisseria meningitidis и способы их применения
WO2013186753A1 (fr) 2012-06-14 2013-12-19 Novartis Ag Vaccins contre le méningocoque de sérogroupe x
JP6324961B2 (ja) 2012-09-06 2018-05-16 ノバルティス アーゲー 血清群ｂ髄膜炎菌とｄ／ｔ／ｐとの組み合わせワクチン
AU2013320313B2 (en) 2012-09-18 2018-07-12 Glaxosmithkline Biologicals Sa Outer membrane vesicles
US20160120818A1 (en) * 2013-02-07 2016-05-05 Glaxosmithline Biological Sa Pharmaceutical compositions comprising vesicles
JP6446377B2 (ja) 2013-03-08 2018-12-26 ファイザー・インク 免疫原性融合ポリペプチド
EP3041502A2 (fr) 2013-09-08 2016-07-13 Pfizer Inc. Compositions utilisables contre neisseria meningitidis et procédés associés
CA2940447C (fr) 2014-02-28 2023-07-11 Glaxosmithkline Biologicals Sa Polypeptides fhbp meningococciques modifies
RU2723045C2 (ru) 2015-02-19 2020-06-08 Пфайзер Инк. Композиции neisseria meningitidis и способы их получения
US12109259B2 (en) 2016-09-02 2024-10-08 Glaxosmithkline Biologicals Sa Vaccines for Neisseria gonorrhoeae
CN108220267B (zh) * 2016-12-22 2022-10-04 丰益(上海)生物技术研发中心有限公司 磷脂酶及其应用
IL267733B2 (en) 2017-01-31 2023-10-01 Pfizer NEISSERIA MENINGITIDIS preparations and methods therefor
KR20200053531A (ko) 2017-09-08 2020-05-18 에벨로 바이오사이언시즈, 인크. 박테리아 세포외 소포
KR20200136361A (ko) 2017-12-04 2020-12-07 드 슈타트 데르 네덜란덴, 베르테겐부어디그트 두어 드 미니스터 반 베이웨이에스, 미니스테리 반 폭스겐트존하이트, 벨지인 엔 스포츠 외막 소포의 생성을 위한 개선된 방법
EP3607967A1 (fr) 2018-08-09 2020-02-12 GlaxoSmithKline Biologicals S.A. Polypeptides modifiés du méningocoque fhbp
CA3126492A1 (fr) * 2019-01-11 2020-07-16 Northwestern University Synthese de vaccins bioconjugues dans des lysats cellulaires procaryotes
BR112021016605A2 (pt) * 2019-02-22 2022-01-18 Evelo Biosciences Inc Preparações de membrana bacteriana
EP3799884A1 (fr) * 2019-10-01 2021-04-07 GlaxoSmithKline Biologicals S.A. Compositions immunogènes
CN111440748A (zh) * 2020-05-15 2020-07-24 黑龙江八一农垦大学 一种分离、提纯及鉴定坏死杆菌外膜囊泡的方法
CA3204887A1 (fr) * 2020-12-11 2022-06-16 Glaxosmithkline Biologicals Sa Bacteries a hyper-boursouflure
GB202115077D0 (en) 2021-10-21 2021-12-08 Glaxosmithkline Biologicals Sa Assay
WO2023097652A1 (fr) * 2021-12-03 2023-06-08 National Center For Nanoscience And Technology Cellule modifiée et son application
GB202211033D0 (en) 2022-07-28 2022-09-14 Glaxosmithkline Biologicals Sa Purification process

Citations (82)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0109942A2 (fr) 1982-10-18 1984-05-30 Bror Morein Complexe protéinique ou peptidique immunogène, procédé pour préparer ce complexe et son usage comme immunostimulant et comme vaccin
EP0301992A2 (fr) 1987-07-30 1989-02-01 Centro Nacional De Biopreparados Vaccin contre Neisséria meningitidis groupe B, gammaglobuline et facteur de transfert
EP0372501A2 (fr) 1988-12-07 1990-06-13 BEHRINGWERKE Aktiengesellschaft Antigènes synthétiques, procédé pour leur préparation et leur utilisation
WO1990006696A2 (fr) 1988-12-19 1990-06-28 Praxis Biologics, Inc. Vaccin meningocoque de la proteine de la membrane externe de la classe 1
EP0378881A1 (fr) 1989-01-17 1990-07-25 ENIRICERCHE S.p.A. Peptides synthétiques et leur utilisation comme support universel pour la préparation de conjugués immunogènes convenant au développement de vaccins synthétiques
WO1990014837A1 (fr) 1989-05-25 1990-12-13 Chiron Corporation Composition d'adjuvant comprenant une emulsion de gouttelettes d'huile d'une taille inferieure au micron
WO1991001146A1 (fr) 1989-07-14 1991-02-07 Praxis Biologics, Inc. Cytokine et supports d'hormone pour vaccins conjugues
EP0427347A1 (fr) 1989-11-10 1991-05-15 ENIRICERCHE S.p.A. Peptides synthétiques utiles comme porteurs universels pour la préparation des conjugués immunogéniques el leur emploi dans le développment des vaccins synthétiques
US5057540A (en) 1987-05-29 1991-10-15 Cambridge Biotech Corporation Saponin adjuvant
EP0471177A2 (fr) 1990-08-13 1992-02-19 American Cyanamid Company Hemagglutinine filamenteuse de Bordetella pertussis à titre de molécules porteuses pour vaccins conjugués
WO1993003761A1 (fr) 1991-08-15 1993-03-04 Board Of Regents, The University Of Texas System PROCEDES ET COMPOSITIONS RELATIFS A DES ANTIGENES UTILES DE $i(MORAXELLA CATARRHALIS)
WO1993017712A2 (fr) 1992-03-06 1993-09-16 Biocine Spa Composes conjugues obtenus a partir de proteines du choc thermique et d'oligosaccharides ou de polysaccharides
WO1994000153A1 (fr) 1992-06-25 1994-01-06 Smithkline Beecham Biologicals (S.A.) Composition vaccinale contenant des adjuvants
WO1994003208A1 (fr) 1992-07-30 1994-02-17 Yeda Research And Development Company Ltd. Conjugues d'antigenes faiblement immunogenes et porteurs de peptides synthetiques et vaccins les contenant
WO1994008021A1 (fr) 1992-10-02 1994-04-14 De Staat Der Nederlanden, Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur Lipopolysaccharide meningococcique immunogenique ainsi que vesicules de membrane externe et vaccins obtenus
EP0594610A1 (fr) 1990-05-31 1994-05-04 Arne Forsgren PROTEINE D - PROTEINE FIXATRICE D'IgD, DE HAEMOPHILUS INFLUENZAE
EP0624376A1 (fr) 1993-05-13 1994-11-17 American Cyanamid Company Préparation et utilisations de protéines de membranes externes dépouvues de LOS à partir de coques gram-négatifs
EP0626169A2 (fr) 1988-08-25 1994-11-30 The Liposome Company, Inc. Form de dosage comprenant un antigen et un dérivé de sel acide d'un stérol
WO1995017211A1 (fr) 1993-12-22 1995-06-29 Biocine S.P.A. Adjuvant non toxique pour les muqueuses
EP0689454A1 (fr) 1993-03-23 1996-01-03 Smithkline Beecham Biolog Compositions vaccinales renfermant le lipide a monophosphorylique 3-o desacetyle
WO1996011711A1 (fr) 1994-10-12 1996-04-25 Iscotec Ab Preparations a base de saponine et leur utilisation dans des complexes immunostimulants
EP0735898A1 (fr) 1993-12-23 1996-10-09 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccins
WO1996033739A1 (fr) 1995-04-25 1996-10-31 Smithkline Beecham Biologicals S.A. Vaccins contenant une saponine ainsi qu'un sterol
WO1997005899A2 (fr) 1995-08-04 1997-02-20 University Of Guelph Nouveaux vaccins et nouvelles compositions pharmaceutiques utilisant des vesicules de membrane de micro-organismes et leurs procedes d'elaboration
EP0835318A2 (fr) 1995-06-29 1998-04-15 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccins contre l'hepatite c
WO1998040100A1 (fr) 1997-03-10 1998-09-17 Ottawa Civic Loeb Research Institute UTILISATION D'ACIDES NUCLEIQUES CONTENANT UN DINUCLEOTIDE CpG NON METHYLE EN TANT QU'ADJUVANT
WO1998042375A1 (fr) 1997-03-21 1998-10-01 Chiron Corporation Mutants detoxiques de toxines bacteriennes ribolysant de l'adp utilises comme adjuvants parenteraux
WO1998056901A2 (fr) 1997-06-09 1998-12-17 Medical Research Council Vaccin vivant attenue
WO1998057659A1 (fr) 1997-06-14 1998-12-23 Smithkline Beecham Biologicals S.A. Compositions adjuvantes destinees a des vaccins
WO1998058668A2 (fr) 1997-06-20 1998-12-30 Microbiological Research Authority VACCIN CONTENANT UN ANTIGENE DE $i(BORDETELLA PERTUSSIS)
WO1999010497A1 (fr) 1997-08-21 1999-03-04 De Staat Der Nederlanden, Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur Nouveaux mutants de bacteries des muqueuses gram negatives et leur application dans des vaccins
WO1999011241A1 (fr) 1997-09-05 1999-03-11 Smithkline Beecham Biologicals S.A. Emulsions huile-dans-l'eau contenant des saponines
WO1999027105A2 (fr) 1997-11-21 1999-06-03 Genset Sequence genomique et polypeptides de chlamydia pneumoniae, leurs fragments et leurs utilisations, en particulier pour le diagnostic, la prevention ou le traitement d'une infection
WO1999027960A1 (fr) 1997-11-28 1999-06-10 West Pharmaceutical Services Compositions vaccinales destinees a etre administrees dans les muqueuses et renfermant un chitosane
WO1999028475A2 (fr) 1997-11-28 1999-06-10 Genset Sequence genomique et polypeptides de chlamydia trachomatis, leurs fragments et leurs utilisations, en particulier, pour le diagnostic, la prevention et le traitement de l'infection
US5916588A (en) 1984-04-12 1999-06-29 The Liposome Company, Inc. Peptide-containing liposomes, immunogenic liposomes and methods of preparation and use
WO1999040936A2 (fr) 1998-02-12 1999-08-19 American Cyanamid Company Vaccins pneumococciques ou meningococciques formules avec l'interleukine-12
WO1999044636A2 (fr) 1998-03-05 1999-09-10 The Medical College Of Ohio Renforcement des reponses immunitaires vis a vis d'antigenes independants de t
WO1999052549A1 (fr) 1998-04-09 1999-10-21 Smithkline Beecham Biologicals S.A. Compositions adjuvantes
US5981213A (en) 1991-08-15 1999-11-09 Board Of Regents, The University Of Texas System Methods and compositions relating to useful antigens of moraxella catarrhalis
WO1999059625A1 (fr) 1998-05-19 1999-11-25 University Of Maryland Systeme d'apport de vaccin
WO1999062923A2 (fr) 1998-06-05 1999-12-09 Dynavax Technologies Corporation Oligonucleotides immunostimulateurs avec bases modifiees et leurs methodes d'utilisation
WO2000007621A2 (fr) 1998-08-05 2000-02-17 Smithkline Beecham Biologicals S.A. Vaccin
WO2000023105A2 (fr) 1998-10-16 2000-04-27 Smithkline Beecham Biologicals S.A. Produits d'addition et vaccins
WO2000025811A2 (fr) 1998-11-02 2000-05-11 Microbiological Research Authority Camr (Centre For Applied Microbiology & Research) Vaccin meningococcique multicomposant
WO2000027994A2 (fr) 1998-11-12 2000-05-18 The Regents Of The University Of California Sequence genomique de chlamydia pneumoniae
WO2000037494A2 (fr) 1998-12-18 2000-06-29 Chiron S.P.A. Antigenes a chlamydia trachomatis
US6090406A (en) 1984-04-12 2000-07-18 The Liposome Company, Inc. Potentiation of immune responses with liposomal adjuvants
WO2000050074A2 (fr) 1999-02-22 2000-08-31 Microbiological Research Authority Compositions de vaccin a neisseria et procedes correspondants
WO2000056360A2 (fr) 1999-03-19 2000-09-28 Smithkline Beecham Biologicals S.A. Vaccin
WO2000061761A2 (fr) 1999-04-09 2000-10-19 Techlab, Inc. Support proteique recombinant de la toxine a pour vaccins conjugues polysaccharides
WO2001009350A2 (fr) 1999-08-03 2001-02-08 Smithkline Beecham Biologicals S.A. Composition de vaccin
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
WO2001021207A2 (fr) 1999-09-24 2001-03-29 Smithkline Beecham Biologicals S.A. Vaccins
WO2001021152A1 (fr) 1999-09-24 2001-03-29 Smithkline Beecham Biologicals S.A. Adjuvant comprenant un ether ou ester d'alkyle polyethylene et au moins un tensioactif non ionique
WO2001034642A2 (fr) 1999-11-12 2001-05-17 University Of Iowa Research Foundation Commande de la synthese de membrane de neisseria
US6239116B1 (en) 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
WO2001052885A1 (fr) 2000-01-17 2001-07-26 Chiron Spa Vaccin a base de vesicule de membrane externe (omv) comprenant des proteines de membrane externe du serogroupe b de n. meningitis
WO2001064922A2 (fr) 2000-02-28 2001-09-07 Chiron Spa Expression heterologue de proteines issues du gonocoque
WO2001072337A1 (fr) 2000-03-27 2001-10-04 Microbiological Research Authority Proteines utilisees comme transporteuses dans des vaccins conjugues
WO2001091788A1 (fr) 2000-06-02 2001-12-06 Statens Institutt For Folkehelse Vaccin comprenant des vesicules de membrane externe utilise contre les maladies causees par le serogroupe a de neisseria meningitidis et methode de preparation
WO2001095935A1 (fr) 2000-01-20 2001-12-20 Ottawa Health Research Institute Acides nucleiques immunostimulateurs permettant d'induire une reponse immunitaire th2
WO2002002606A2 (fr) 2000-07-03 2002-01-10 Chiron S.P.A. Immunisation contre une infection par chlamydia pneumoniae
WO2002007763A2 (fr) 2000-07-12 2002-01-31 Chiron S.P.A. Helicobacter pylori avec biosynthese de lipopolysaccharides modifies
WO2002009643A2 (fr) 2000-07-27 2002-02-07 Children's Hospital & Research Center At Oakland Vaccins pour protection a large spectre contre les maladies causees par neisseria meningitidis
WO2002009746A2 (fr) 2000-07-31 2002-02-07 Glaxosmithkline Biologicals S.A. Composition vaccinale
WO2002026757A2 (fr) 2000-09-26 2002-04-04 Hybridon, Inc. Modulation de l'activite immunostimulatrice d'analogues oligonucleotidiques immunostimulateurs par des modifications chimiques de position
WO2002034771A2 (fr) 2000-10-27 2002-05-02 Chiron Srl Acides nucleiques et proteines derives des groupes de streptocoques a et b
US6429199B1 (en) 1994-07-15 2002-08-06 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules for activating dendritic cells
WO2002062378A2 (fr) 2001-02-08 2002-08-15 Smithkline Beecham Biologicals S.A. Composition de vaccin
WO2002079243A2 (fr) 2001-02-12 2002-10-10 Chiron Srl. Proteines gonococciques et acides nucleiques
WO2002091998A2 (fr) 2001-05-11 2002-11-21 Aventis Pasteur, Inc. Nouveau vaccin conjugue contre la meningite
WO2003007985A2 (fr) 2001-06-20 2003-01-30 Chiron Srl. Solubilisation de polysaccharides capsulaires et vaccins de combinaison
WO2003009869A1 (fr) 2001-07-26 2003-02-06 Chiron Srl. Vaccins comprenant des adjuvants aluminium et histidine
WO2003024481A2 (fr) 2001-09-14 2003-03-27 Cytos Biotechnology Ag Emballage de substances immunnostimulatrices dans des particules de type virus : procede de preparation et utilisation correspondants
WO2003024480A2 (fr) 2001-09-14 2003-03-27 Cytos Biotechnology Ag Activation in vivo de cellules presentant un antigene en vue d'augmenter les reponses immunes induites par des particules de type virus
WO2003035836A2 (fr) 2001-10-24 2003-05-01 Hybridon Inc. Modulation des proprietes immunostimulantes de composes a base d'oligonucleotides par presentation optimale d'extremites 5'
WO2004014417A2 (fr) 2002-08-02 2004-02-19 Glaxosmithkline Biologicals Sa Vaccins
WO2004019977A2 (fr) 2002-08-30 2004-03-11 Chiron Srl Ameliorations apportees a des vesicules de membrane externe bacteriennes
WO2004046177A2 (fr) 2002-11-15 2004-06-03 Chiron Srl Proteines de surface inattendues sur le meningocoque
WO2004048404A2 (fr) 2002-11-22 2004-06-10 Chiron Srl Variants multiples de la proteine nmb1870 meningococcique
WO2006046143A2 (fr) 2004-10-29 2006-05-04 Novartis Vaccines And Diagnostics Srl Vesicules bacteriennes immunogenes comprenant des proteines de la membrane externe

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2848965A1 (de) 1978-11-11 1980-05-22 Behringwerke Ag Verfahren zur herstellung von membranproteinen aus neisseria meningitidis und diese enthaltende vaccine
WO1995029662A2 (fr) 1994-04-20 1995-11-09 U.S. Department Of The Army Vaccin contre les infections par des bacteries gram-negatives
KR20010021953A (ko) 1997-07-17 2001-03-15 추후제출 Ｂ군 수막염균 포린 및 에이치.인플루엔자 다당류를포함하는 면역원성 접합체
US8007815B1 (en) 1998-05-29 2011-08-30 Novartis Ag Combination meningitidis B/C vaccines
EP1741443B1 (fr) 1998-05-29 2014-05-21 Novartis Vaccines and Diagnostics, Inc. Vaccin B/C combiné contre la méningite
GB0118249D0 (en) 2001-07-26 2001-09-19 Chiron Spa Histidine vaccines
AR045702A1 (es) 2001-10-03 2005-11-09 Chiron Corp Composiciones de adyuvantes.
GB0130123D0 (en) 2001-12-17 2002-02-06 Microbiological Res Agency Outer membrane vesicle vaccine and its preparation
CA2501812C (fr) 2002-10-11 2012-07-10 Mariagrazia Pizza Vaccins polypeptidiques offrant une large protection contre des lignees de meningocoques hypervirulentes
AU2003288558A1 (en) 2002-12-16 2004-07-09 Nasjonalt Folkehelseinstitutt Meningococcal vaccine based on outer membrane proteins porb2 and pora
GB0316560D0 (en) 2003-07-15 2003-08-20 Chiron Srl Vesicle filtration
JP5173194B2 (ja) 2003-12-23 2013-03-27 グラクソスミスクライン バイオロジカルズ ソシエテ アノニム ワクチン
GB0419627D0 (en) 2004-09-03 2004-10-06 Chiron Srl Immunogenic bacterial vesicles with outer membrane proteins
HUE033196T2 (en) 2005-01-27 2017-11-28 Children's Hospital & Res Center At Oakland GNA1870-based vesicle vaccines for broad-spectrum protection against diseases caused by Neisseria meningitidis
AU2009262893B2 (en) 2008-05-30 2015-05-21 The U.S.A., as represented by The Secretary of the Army, on behalf of Walter Reed Army Institute Of Research Meningococcal multivalent native outer membrane vesicle vaccine, methods of making and use thereof
GB0816284D0 (en) 2008-09-05 2008-10-15 Nat Biolog Standards Board Vaccine

Patent Citations (90)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0109942A2 (fr) 1982-10-18 1984-05-30 Bror Morein Complexe protéinique ou peptidique immunogène, procédé pour préparer ce complexe et son usage comme immunostimulant et comme vaccin
US6090406A (en) 1984-04-12 2000-07-18 The Liposome Company, Inc. Potentiation of immune responses with liposomal adjuvants
US5916588A (en) 1984-04-12 1999-06-29 The Liposome Company, Inc. Peptide-containing liposomes, immunogenic liposomes and methods of preparation and use
US5057540A (en) 1987-05-29 1991-10-15 Cambridge Biotech Corporation Saponin adjuvant
US5747653A (en) 1987-07-30 1998-05-05 Centro Nacional De Biopreparados Method of producing of an antimeningococcic hyperimmune gamma globulin and gamma globulin produced by method
EP0301992A2 (fr) 1987-07-30 1989-02-01 Centro Nacional De Biopreparados Vaccin contre Neisséria meningitidis groupe B, gammaglobuline et facteur de transfert
US5597572A (en) 1987-07-30 1997-01-28 Centro Nacional De Biopreparados Method of producing Neisseria meningitidis B vaccine, and vaccine produced by method
EP0626169A2 (fr) 1988-08-25 1994-11-30 The Liposome Company, Inc. Form de dosage comprenant un antigen et un dérivé de sel acide d'un stérol
EP0372501A2 (fr) 1988-12-07 1990-06-13 BEHRINGWERKE Aktiengesellschaft Antigènes synthétiques, procédé pour leur préparation et leur utilisation
WO1990006696A2 (fr) 1988-12-19 1990-06-28 Praxis Biologics, Inc. Vaccin meningocoque de la proteine de la membrane externe de la classe 1
EP0449958A1 (fr) 1988-12-19 1991-10-09 Praxis Biolog Inc Vaccin meningocoque de la proteine de la membrane externe de la classe 1.
EP0378881A1 (fr) 1989-01-17 1990-07-25 ENIRICERCHE S.p.A. Peptides synthétiques et leur utilisation comme support universel pour la préparation de conjugués immunogènes convenant au développement de vaccins synthétiques
WO1990014837A1 (fr) 1989-05-25 1990-12-13 Chiron Corporation Composition d'adjuvant comprenant une emulsion de gouttelettes d'huile d'une taille inferieure au micron
WO1991001146A1 (fr) 1989-07-14 1991-02-07 Praxis Biologics, Inc. Cytokine et supports d'hormone pour vaccins conjugues
EP0427347A1 (fr) 1989-11-10 1991-05-15 ENIRICERCHE S.p.A. Peptides synthétiques utiles comme porteurs universels pour la préparation des conjugués immunogéniques el leur emploi dans le développment des vaccins synthétiques
EP0594610A1 (fr) 1990-05-31 1994-05-04 Arne Forsgren PROTEINE D - PROTEINE FIXATRICE D'IgD, DE HAEMOPHILUS INFLUENZAE
EP0471177A2 (fr) 1990-08-13 1992-02-19 American Cyanamid Company Hemagglutinine filamenteuse de Bordetella pertussis à titre de molécules porteuses pour vaccins conjugués
WO1993003761A1 (fr) 1991-08-15 1993-03-04 Board Of Regents, The University Of Texas System PROCEDES ET COMPOSITIONS RELATIFS A DES ANTIGENES UTILES DE $i(MORAXELLA CATARRHALIS)
US5981213A (en) 1991-08-15 1999-11-09 Board Of Regents, The University Of Texas System Methods and compositions relating to useful antigens of moraxella catarrhalis
US5552146A (en) 1991-08-15 1996-09-03 Board Of Regents, The University Of Texas System Methods and compositions relating to useful antigens of Moraxella catarrhalis
WO1993017712A2 (fr) 1992-03-06 1993-09-16 Biocine Spa Composes conjugues obtenus a partir de proteines du choc thermique et d'oligosaccharides ou de polysaccharides
WO1994000153A1 (fr) 1992-06-25 1994-01-06 Smithkline Beecham Biologicals (S.A.) Composition vaccinale contenant des adjuvants
EP0761231A1 (fr) 1992-06-25 1997-03-12 SMITHKLINE BEECHAM BIOLOGICALS s.a. Composition vaccinale contenant des adjuvants
WO1994003208A1 (fr) 1992-07-30 1994-02-17 Yeda Research And Development Company Ltd. Conjugues d'antigenes faiblement immunogenes et porteurs de peptides synthetiques et vaccins les contenant
US5705161A (en) 1992-10-02 1998-01-06 De Staat Der Nederlanden, Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur Immunogenic meningococcal LPS and other membrane vesicles and vaccine therefrom
WO1994008021A1 (fr) 1992-10-02 1994-04-14 De Staat Der Nederlanden, Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur Lipopolysaccharide meningococcique immunogenique ainsi que vesicules de membrane externe et vaccins obtenus
US5993826A (en) 1993-03-02 1999-11-30 Board Of Regents, The University Of Texas Methods and compositions relating to useful antigens of moraxella catarrhalis
EP0689454A1 (fr) 1993-03-23 1996-01-03 Smithkline Beecham Biolog Compositions vaccinales renfermant le lipide a monophosphorylique 3-o desacetyle
EP0624376A1 (fr) 1993-05-13 1994-11-17 American Cyanamid Company Préparation et utilisations de protéines de membranes externes dépouvues de LOS à partir de coques gram-négatifs
WO1995017211A1 (fr) 1993-12-22 1995-06-29 Biocine S.P.A. Adjuvant non toxique pour les muqueuses
EP0735898A1 (fr) 1993-12-23 1996-10-09 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccins
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6239116B1 (en) 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6429199B1 (en) 1994-07-15 2002-08-06 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules for activating dendritic cells
WO1996011711A1 (fr) 1994-10-12 1996-04-25 Iscotec Ab Preparations a base de saponine et leur utilisation dans des complexes immunostimulants
WO1996033739A1 (fr) 1995-04-25 1996-10-31 Smithkline Beecham Biologicals S.A. Vaccins contenant une saponine ainsi qu'un sterol
US6180111B1 (en) 1995-05-18 2001-01-30 University Of Maryland Vaccine delivery system
EP0835318A2 (fr) 1995-06-29 1998-04-15 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccins contre l'hepatite c
WO1997005899A2 (fr) 1995-08-04 1997-02-20 University Of Guelph Nouveaux vaccins et nouvelles compositions pharmaceutiques utilisant des vesicules de membrane de micro-organismes et leurs procedes d'elaboration
WO1998040100A1 (fr) 1997-03-10 1998-09-17 Ottawa Civic Loeb Research Institute UTILISATION D'ACIDES NUCLEIQUES CONTENANT UN DINUCLEOTIDE CpG NON METHYLE EN TANT QU'ADJUVANT
WO1998042375A1 (fr) 1997-03-21 1998-10-01 Chiron Corporation Mutants detoxiques de toxines bacteriennes ribolysant de l'adp utilises comme adjuvants parenteraux
WO1998056901A2 (fr) 1997-06-09 1998-12-17 Medical Research Council Vaccin vivant attenue
WO1998057659A1 (fr) 1997-06-14 1998-12-23 Smithkline Beecham Biologicals S.A. Compositions adjuvantes destinees a des vaccins
WO1998058668A2 (fr) 1997-06-20 1998-12-30 Microbiological Research Authority VACCIN CONTENANT UN ANTIGENE DE $i(BORDETELLA PERTUSSIS)
WO1999010497A1 (fr) 1997-08-21 1999-03-04 De Staat Der Nederlanden, Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur Nouveaux mutants de bacteries des muqueuses gram negatives et leur application dans des vaccins
WO1999011241A1 (fr) 1997-09-05 1999-03-11 Smithkline Beecham Biologicals S.A. Emulsions huile-dans-l'eau contenant des saponines
WO1999027105A2 (fr) 1997-11-21 1999-06-03 Genset Sequence genomique et polypeptides de chlamydia pneumoniae, leurs fragments et leurs utilisations, en particulier pour le diagnostic, la prevention ou le traitement d'une infection
WO1999028475A2 (fr) 1997-11-28 1999-06-10 Genset Sequence genomique et polypeptides de chlamydia trachomatis, leurs fragments et leurs utilisations, en particulier, pour le diagnostic, la prevention et le traitement de l'infection
WO1999027960A1 (fr) 1997-11-28 1999-06-10 West Pharmaceutical Services Compositions vaccinales destinees a etre administrees dans les muqueuses et renfermant un chitosane
WO1999040936A2 (fr) 1998-02-12 1999-08-19 American Cyanamid Company Vaccins pneumococciques ou meningococciques formules avec l'interleukine-12
WO1999044636A2 (fr) 1998-03-05 1999-09-10 The Medical College Of Ohio Renforcement des reponses immunitaires vis a vis d'antigenes independants de t
WO1999052549A1 (fr) 1998-04-09 1999-10-21 Smithkline Beecham Biologicals S.A. Compositions adjuvantes
WO1999059625A1 (fr) 1998-05-19 1999-11-25 University Of Maryland Systeme d'apport de vaccin
WO1999062923A2 (fr) 1998-06-05 1999-12-09 Dynavax Technologies Corporation Oligonucleotides immunostimulateurs avec bases modifiees et leurs methodes d'utilisation
WO2000007621A2 (fr) 1998-08-05 2000-02-17 Smithkline Beecham Biologicals S.A. Vaccin
WO2000023105A2 (fr) 1998-10-16 2000-04-27 Smithkline Beecham Biologicals S.A. Produits d'addition et vaccins
WO2000025811A2 (fr) 1998-11-02 2000-05-11 Microbiological Research Authority Camr (Centre For Applied Microbiology & Research) Vaccin meningococcique multicomposant
WO2000027994A2 (fr) 1998-11-12 2000-05-18 The Regents Of The University Of California Sequence genomique de chlamydia pneumoniae
WO2000037494A2 (fr) 1998-12-18 2000-06-29 Chiron S.P.A. Antigenes a chlamydia trachomatis
WO2000050074A2 (fr) 1999-02-22 2000-08-31 Microbiological Research Authority Compositions de vaccin a neisseria et procedes correspondants
WO2000056360A2 (fr) 1999-03-19 2000-09-28 Smithkline Beecham Biologicals S.A. Vaccin
WO2000061761A2 (fr) 1999-04-09 2000-10-19 Techlab, Inc. Support proteique recombinant de la toxine a pour vaccins conjugues polysaccharides
WO2001009350A2 (fr) 1999-08-03 2001-02-08 Smithkline Beecham Biologicals S.A. Composition de vaccin
WO2001021152A1 (fr) 1999-09-24 2001-03-29 Smithkline Beecham Biologicals S.A. Adjuvant comprenant un ether ou ester d'alkyle polyethylene et au moins un tensioactif non ionique
WO2001021207A2 (fr) 1999-09-24 2001-03-29 Smithkline Beecham Biologicals S.A. Vaccins
WO2001034642A2 (fr) 1999-11-12 2001-05-17 University Of Iowa Research Foundation Commande de la synthese de membrane de neisseria
WO2001052885A1 (fr) 2000-01-17 2001-07-26 Chiron Spa Vaccin a base de vesicule de membrane externe (omv) comprenant des proteines de membrane externe du serogroupe b de n. meningitis
WO2001095935A1 (fr) 2000-01-20 2001-12-20 Ottawa Health Research Institute Acides nucleiques immunostimulateurs permettant d'induire une reponse immunitaire th2
WO2001064922A2 (fr) 2000-02-28 2001-09-07 Chiron Spa Expression heterologue de proteines issues du gonocoque
WO2001072337A1 (fr) 2000-03-27 2001-10-04 Microbiological Research Authority Proteines utilisees comme transporteuses dans des vaccins conjugues
WO2001091788A1 (fr) 2000-06-02 2001-12-06 Statens Institutt For Folkehelse Vaccin comprenant des vesicules de membrane externe utilise contre les maladies causees par le serogroupe a de neisseria meningitidis et methode de preparation
WO2002002606A2 (fr) 2000-07-03 2002-01-10 Chiron S.P.A. Immunisation contre une infection par chlamydia pneumoniae
WO2002007763A2 (fr) 2000-07-12 2002-01-31 Chiron S.P.A. Helicobacter pylori avec biosynthese de lipopolysaccharides modifies
WO2002009643A2 (fr) 2000-07-27 2002-02-07 Children's Hospital & Research Center At Oakland Vaccins pour protection a large spectre contre les maladies causees par neisseria meningitidis
WO2002009746A2 (fr) 2000-07-31 2002-02-07 Glaxosmithkline Biologicals S.A. Composition vaccinale
WO2002026757A2 (fr) 2000-09-26 2002-04-04 Hybridon, Inc. Modulation de l'activite immunostimulatrice d'analogues oligonucleotidiques immunostimulateurs par des modifications chimiques de position
WO2002034771A2 (fr) 2000-10-27 2002-05-02 Chiron Srl Acides nucleiques et proteines derives des groupes de streptocoques a et b
WO2002062378A2 (fr) 2001-02-08 2002-08-15 Smithkline Beecham Biologicals S.A. Composition de vaccin
WO2002079243A2 (fr) 2001-02-12 2002-10-10 Chiron Srl. Proteines gonococciques et acides nucleiques
WO2002091998A2 (fr) 2001-05-11 2002-11-21 Aventis Pasteur, Inc. Nouveau vaccin conjugue contre la meningite
WO2003007985A2 (fr) 2001-06-20 2003-01-30 Chiron Srl. Solubilisation de polysaccharides capsulaires et vaccins de combinaison
WO2003009869A1 (fr) 2001-07-26 2003-02-06 Chiron Srl. Vaccins comprenant des adjuvants aluminium et histidine
WO2003024481A2 (fr) 2001-09-14 2003-03-27 Cytos Biotechnology Ag Emballage de substances immunnostimulatrices dans des particules de type virus : procede de preparation et utilisation correspondants
WO2003024480A2 (fr) 2001-09-14 2003-03-27 Cytos Biotechnology Ag Activation in vivo de cellules presentant un antigene en vue d'augmenter les reponses immunes induites par des particules de type virus
WO2003035836A2 (fr) 2001-10-24 2003-05-01 Hybridon Inc. Modulation des proprietes immunostimulantes de composes a base d'oligonucleotides par presentation optimale d'extremites 5'
WO2004014417A2 (fr) 2002-08-02 2004-02-19 Glaxosmithkline Biologicals Sa Vaccins
WO2004019977A2 (fr) 2002-08-30 2004-03-11 Chiron Srl Ameliorations apportees a des vesicules de membrane externe bacteriennes
WO2004046177A2 (fr) 2002-11-15 2004-06-03 Chiron Srl Proteines de surface inattendues sur le meningocoque
WO2004048404A2 (fr) 2002-11-22 2004-06-10 Chiron Srl Variants multiples de la proteine nmb1870 meningococcique
WO2006046143A2 (fr) 2004-10-29 2006-05-04 Novartis Vaccines And Diagnostics Srl Vesicules bacteriennes immunogenes comprenant des proteines de la membrane externe

Non-Patent Citations (99)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Molecular Biology", vol. 30, 1987
"Vaccine Design.", 1995, PLENUM
"Vaccines", 2004
ADU-BOBIE ET AL., INFECT IMMUN, vol. 72, 2004, pages 1914 - 1919
ADU-BOBIE JEANNETTE ET AL: "GNA33 of Neisseria meningitidis is a lipoprotein required for cell separation, membrane architecture, and virulence", INFECTION AND IMMUNITY, vol. 72, no. 4, April 2004 (2004-04-01), pages 1914 - 1919, XP001189657, ISSN: 0019-9567 *
AGARWAL; MISHRA, INDIAN JEXP BIOL, vol. 37, 1999, pages 6 - 16
ALMEIDA; ALPAR, J. DRUG TARGETING, vol. 3, 1996, pages 455 - 467
ANDERSON ET AL., J CLIN INVEST, vol. 76, no. 1, 1985, pages 52 - 59
ANDERSON, INFECT IMMUN, vol. 39, no. 1, 1983, pages 233 - 238
ANDRIANOV ET AL., BIOMATERIALS, vol. 19, 1998, pages 109 - 115
ANONYMOUS, RESEARCH DISCLOSURE, January 2002 (2002-01-01), pages 453077
ARIGITA ET AL., VACCINE, vol. 21, 2003, pages 950 - 960
BAKKE ET AL., INFECT. IMMUN., vol. 69, 2001, pages 5010 - 5015
BARR ET AL., ADVANCED DRUG DELIVERY REVIEWS, vol. 32, 1998, pages 247 - 271
BEIGNON ET AL., INFECT IMMUN, vol. 70, 2002, pages 3012 - 3019
BELL, PEDIATR INFECT DIS J, vol. 19, 2000, pages 1187 - 1188
BERNADAC ALAIN ET AL: "Escherichia coli tol-pal mutants form outer membrane vesicles", JOURNAL OF BACTERIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, WASHINGTON, DC; US, vol. 180, no. 18, 1 September 1998 (1998-09-01), pages 4872 - 4878, XP002208245, ISSN: 0021-9193 *
BERNADAC ET AL., JBACTERIOL, vol. 180, no. 18, 1998, pages 4872 - 8
BEVERIDGE, JBACTERIOL, vol. 181, 1999, pages 4725 - 33
BHAGAT ET AL., BBRC, vol. 300, 2003, pages 853 - 861
BJUNE ET AL., LANCET, vol. 338, no. 8775, 1991, pages 1093 - 1096
BLACKWELL ET AL., J LMMUNOL, vol. 170, 2003, pages 4061 - 4068
BLANCO ET AL., J LMMUNOL, vol. 163, 1999, pages 2741 - 2746
COSTANTINO ET AL., VACCINE, vol. 10, 1992, pages 691 - 698
DALE, INFECT DIS CLIN NORTH AM, vol. 13, no. VIII, 1999, pages 227 - 43
DE KLEIJN ET AL., VACCINE, vol. 20, 2001, pages 352 - 358
DOMENIGHINI ET AL., MOL MICROBIOL, vol. 15, 1995, pages 1165 - 1167
DREESEN, VACCINE, vol. 15, 1997, pages 2 - 6
EVANS ET AL., EXPERT REV VACCINES, vol. 2, 2003, pages 219 - 229
FALUGI ET AL., EUR J IMMUNOL, vol. 31, 2001, pages 3816 - 3824
FERRETTI ET AL., PNAS USA, vol. 98, 2001, pages 4658 - 4663
FREDRIKSEN ET AL., NIPHANN, vol. 14, 1991, pages 67 - 79
GENNARO: "Remington: The Science and Practice of Pharmacy", 2000
GERBER ET AL., VIROL, vol. 75, 2001, pages 4752 - 4760
GERLICH ET AL., VACCINE, vol. 8, 1990, pages 63 - 68,79-80
GLUCK ET AL., VACCINE, vol. 20, 2002, pages B10 - B16
GUSTAFSSON ET AL., N. ENGL. J. MED., vol. 334, 1996, pages 349 - 355
GUTHRIE ET AL., INFECT IMMUN, vol. 72, 2004, pages 2528 - 37
HENRY T ET AL: "Improved methods for producing outer membrane vesicles in Gram-negative bacteria", RESEARCH IN MICROBIOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 155, no. 6, 4 May 2004 (2004-05-04), pages 437 - 446, XP002407763, ISSN: 0923-2508, DOI: 10.1016/J.RESMIC.2004.04.007 *
HSU ET AL., CLIN LIVER DIS, vol. 3, 1999, pages 901 - 915
IWARSON, APMIS, vol. 103, 1995, pages 321 - 326
JEDRZEJAS, MICROBIOL MOL BIOL REV, vol. 65, 2001, pages 187 - 207
JENNINGS ET AL., EUR JBIOCHEM, vol. 269, 2002, pages 3722 - 3731
JOHNSON ET AL., BIOORG MED CHEM LETT, vol. 9, 1999, pages 2273 - 2278
JONES, CURR OPIN INVESTIG DRUGS, vol. 4, 2003, pages 214 - 218
KADURUGAMUWA; BEVERIDGE, MICROBIOLOGY, vol. 145, 1999, pages 2051 - 2060
KALMAN ET AL., NATURE GENETICS, vol. 21, 1999, pages 385 - 389
KANDIMALLA ET AL., BBRC, vol. 306, 2003, pages 948 - 953
KANDIMALLA ET AL., BIOCHEMICAL SOCIETY TRANSACTIONS, vol. 31, 2003, pages 654 - 658
KANDIMALLA ET AL., NUCLEIC ACIDS RESEARCH, vol. 31, 2003, pages 2393 - 2400
KATIAL ET AL., INFECT IMMUN, vol. 70, 2002, pages 702 - 7
KEENAN ET AL., FEMS MICROBIOL LETT, vol. 161, 1998, pages 21 - 27
KESAVALU ET AL., INFECT. IMMUN., vol. 60, 1992, pages 1455 - 1464
KRIEG, NATURE MEDICINE, vol. 9, 2003, pages 831 - 835
KRIEG, TRENDS IMMUNOL, vol. 23, 2002, pages 64 - 65
KUO ET AL., INFECT IMMUN, vol. 63, 1995, pages 2706 - 13
LENZ ET AL., J LMMUNOL, vol. 166, 2001, pages 5346 - 5355
LOMMATZSCH J ET AL: "Outer membrane localization of murein hydrolases: MltA, a third lipoprotein lytic transglycosylase in Escherichia coli", JOURNAL OF BACTERIOLOGY, WASHINGTON, DC, US, vol. 179, no. 17, September 1997 (1997-09-01), pages 5465 - 5470, XP002130154, ISSN: 0021-9193 *
MASIGNANI ET AL., JEXP MED, vol. 197, 2003, pages 789 - 799
MCCLUSKIE ET AL., FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY, vol. 32, 2002, pages 179 - 185
MCMICHAEL, VACCINE, vol. 19, no. 1, 2000, pages 5101 - 107
MERALDI ET AL., VACCINE, vol. 21, 2003, pages 2485 - 2491
MMWR MORB MORTAL WKLY REP, vol. 47, no. 1, 16 January 1998 (1998-01-16), pages 12,19
MOE ET AL., INFECT IMMUN, vol. 69, 2001, pages 3762 - 71
MOE ET AL., INFECT IMMUN, vol. 70, 2002, pages 6021 - 31
NIIKURA ET AL., VIROLOGY, vol. 293, 2002, pages 273 - 280
PAJAK ET AL., VACCINE, vol. 21, 2003, pages 836 - 842
PARMAR ET AL., VACCINE, vol. 15, 1997, pages 1641 - 1651
PARTIDOS ET AL., IMMUNOL LETT, vol. 67, 1999, pages 209 - 216
PAYNE ET AL., ADV DRUG DELIVERY REVIEW, vol. 31, 1998, pages 185 - 196
PEPPOLONI ET AL., EXPERT REV VACCINES, vol. 2, 2003, pages 285 - 293
PINE ET AL., J CONTROL RELEASE, vol. 85, 2002, pages 263 - 270
PINTO ET AL., J LNFECT DIS, vol. 188, 2003, pages 327 - 338
PIZZA ET AL., INT JMED MICROBIOL, vol. 290, 2000, pages 455 - 461
PIZZA ET AL., SCIENCE, vol. 287, 2000, pages 1816 - 1820
PIZZA ET AL., VACCINE, vol. 19, 2001, pages 2534 - 2541
POLLARD; MOXON, ARCH DIS CHILD, vol. 87, 2002, pages 13 - 17
RAPPUOLI ET AL., TIBTECH, vol. 9, 1991, pages 232 - 238
READ ET AL., NUCLEIC ACIDS RES, vol. 28, 2000, pages 1397 - 406
ROSS ET AL., VACCINE, vol. 19, 2001, pages 4135 - 4142
RUBIN, PEDIATR CLIN NORTH AM, vol. 47, 2000, pages 269 - 285
RUSSO; JOHNSON, J LNFECT DIS, vol. 181, 2000, pages 1753 - 4
RYAN ET AL., INFECT IMMUN, vol. 67, 1999, pages 6270 - 6280
SCHARTON-KERSTEN ET AL., INFECT IMMUN, vol. 68, 2000, pages 5306 - 5313
SCHUCHAT, LANCET, vol. 353, no. 9146, 1999, pages 51 - 6
SEIB ET AL., FEBS LETT, vol. 546, 2003, pages 411 - 5
SHIRAI ET AL., J. INFECT. DIS., vol. 181, no. 3, 2000, pages 5524 - 5527
SHOCKMAN; HOLTJE: "Bacterial Cell Wall", 1994, article "Microbial peptidoglycan (murein) hydrolases", pages: 131 - 166
SINGH ET AL., J CONT RELEAS, vol. 70, 2001, pages 267 - 276
SJOLANDER ET AL., ADVANCED DRUG DELIVERY REVIEWS, vol. 32, 1998, pages 321 - 338
SMITH; WATERMAN, ADV. APPL. MATH., vol. 2, 1981, pages 482 - 489
STANLEY, CLIN EXP DERMATOL, vol. 27, 2002, pages 571 - 577
STEEGHS ET AL., THE EMBO JOURNAL, vol. 20, 2001, pages 6937 - 6945
SUTTER ET AL., PEDIATR CLIN NORTH AM, vol. 47, 2000, pages 287 - 308
VAN DER LEY ET AL., VACCINE, vol. 13, 1995, pages 401 - 7
WATSON, PEDIATR INFECT DIS J, vol. 19, 2000, pages 331 - 332
ZHOU ET AL., FEMSMICROBIOL LETT, vol. 163, 1998, pages 223 - 228
ZHU ET AL., JBACTERIOL, vol. 182, 2000, pages 439 - 47
ZIMMERMAN; SPANN, AM FAM PHYSICIAN, vol. 59, 1999, pages 113 - 118,125-126

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3263695A1 (fr) 2016-06-29 2018-01-03 GlaxoSmithKline Biologicals SA Compositions immunogènes
WO2018002270A1 (fr) 2016-06-29 2018-01-04 Glaxosmithkline Biologicals S.A. Compositions immunogènes

Also Published As

Publication number Publication date
BRPI0517514A (pt) 2008-10-14
JP2016135139A (ja) 2016-07-28
CA2584778C (fr) 2014-04-08
US10336794B2 (en) 2019-07-02
US20100015212A1 (en) 2010-01-21
JP5362221B2 (ja) 2013-12-11
JP2011188870A (ja) 2011-09-29
US9771399B2 (en) 2017-09-26
GB0424092D0 (en) 2004-12-01
EP2279747B1 (fr) 2014-06-25
PT2279747E (pt) 2014-10-02
WO2006046143A2 (fr) 2006-05-04
US20160108094A1 (en) 2016-04-21
AU2005298332B2 (en) 2012-05-03
US20180009852A1 (en) 2018-01-11
WO2006046143A3 (fr) 2007-03-15
EP1804834B1 (fr) 2011-05-25
JP2008517617A (ja) 2008-05-29
ATE510559T1 (de) 2011-06-15
JP2014039566A (ja) 2014-03-06
CA2584778A1 (fr) 2006-05-04
US9206399B2 (en) 2015-12-08
CY1112105T1 (el) 2015-11-04
CN101048175A (zh) 2007-10-03
NZ553989A (en) 2010-03-26
JP6077983B2 (ja) 2017-02-08
BRPI0517514A8 (pt) 2017-10-03
ES2507499T3 (es) 2014-10-15
CN101048175B (zh) 2013-03-13
PL2279747T3 (pl) 2015-01-30
AU2005298332A1 (en) 2006-05-04
EP1804834A2 (fr) 2007-07-11

Similar Documents

Publication Publication Date Title
US10336794B2 (en) Immunogenic bacterial vesicles with outer membrane proteins
EP1784213B1 (fr) Ameliorations en rapport avec les vesicules de la membrane exterieure de meningocoque
EP2245048B1 (fr) Polypeptides fhbp à méningocoques
US8334114B2 (en) Lactoferrin cleavage of neisserial proteins
AU2012211495B2 (en) Immunogenic bacterial vesicles with outer membrane proteins
MX2007004035A (es) Vesciculas bacterianas inmunogenicas con proteinas de membrana externa

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AC Divisional application: reference to earlier application

Ref document number: 1804834

Country of ref document: EP

Kind code of ref document: P

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA HR MK YU

17P Request for examination filed

Effective date: 20110802

17Q First examination report despatched

Effective date: 20111206

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

INTG Intention to grant announced

Effective date: 20140109

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AC Divisional application: reference to earlier application

Ref document number: 1804834

Country of ref document: EP

Kind code of ref document: P

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA HR MK YU

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

Ref country code: CH

Ref legal event code: NV

Representative=s name: E. BLUM AND CO. AG PATENT- UND MARKENANWAELTE , CH

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 674189

Country of ref document: AT

Kind code of ref document: T

Effective date: 20140715

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602005044043

Country of ref document: DE

Effective date: 20140807

REG Reference to a national code

Ref country code: PT

Ref legal event code: SC4A

Free format text: AVAILABILITY OF NATIONAL TRANSLATION

Effective date: 20140813

REG Reference to a national code

Ref country code: NL

Ref legal event code: T3

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2507499

Country of ref document: ES

Kind code of ref document: T3

Effective date: 20141015

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140926

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140625

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140625

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140625

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140625

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140625

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140625

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140625

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140625

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140625

REG Reference to a national code

Ref country code: PL

Ref legal event code: T3

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141025

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602005044043

Country of ref document: DE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140625

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141028

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140625

26N No opposition filed

Effective date: 20150326

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140625

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140625

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20051028

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 12

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: ISLER AND PEDRAZZINI AG, CH

Ref country code: CH

Ref legal event code: PFA

Owner name: GSK VACCINES S.R.L., IT

Free format text: FORMER OWNER: NOVARTIS VACCINES AND DIAGNOSTICS S.R.L., IT

REG Reference to a national code

Ref country code: DE

Ref legal event code: R082

Ref document number: 602005044043

Country of ref document: DE

Representative=s name: HOFFMANN - EITLE PATENT- UND RECHTSANWAELTE PA, DE

REG Reference to a national code

Ref country code: NL

Ref legal event code: PD

Owner name: GLAXOSMITHKLINE BIOLOGICALS S.A.; BE

Free format text: DETAILS ASSIGNMENT: CHANGE OF OWNER(S), ASSIGNMENT; FORMER OWNER NAME: NOVARTIS VACCINES AND DIAGNOSTICS S.R.L.

Effective date: 20170503

REG Reference to a national code

Ref country code: DE

Ref legal event code: R082

Ref document number: 602005044043

Country of ref document: DE

Representative=s name: HOFFMANN - EITLE PATENT- UND RECHTSANWAELTE PA, DE

Ref country code: DE

Ref legal event code: R081

Ref document number: 602005044043

Country of ref document: DE

Owner name: GLAXOSMITHKLINE BIOLOGICALS S.A., BE

Free format text: FORMER OWNER: NOVARTIS VACCINES AND DIAGNOSTICS S.R.L., SIENA, SI., IT

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 13

REG Reference to a national code

Ref country code: GB

Ref legal event code: 732E

Free format text: REGISTERED BETWEEN 20170921 AND 20170927

REG Reference to a national code

Ref country code: ES

Ref legal event code: PC2A

Owner name: GLAXOSMITHKLINE BIOLOGICALS S.A.

Effective date: 20171205

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 14

REG Reference to a national code

Ref country code: AT

Ref legal event code: PC

Ref document number: 674189

Country of ref document: AT

Kind code of ref document: T

Owner name: GLAXOSMITHKLINE BIOLOGICALS S.A., BE

Effective date: 20180913

REG Reference to a national code

Ref country code: BE

Ref legal event code: FP

Effective date: 20140910

Ref country code: BE

Ref legal event code: PD

Owner name: GLAXOSMITHKLINE BIOLOGICALS SA; BE

Free format text: DETAILS ASSIGNMENT: CHANGE OF OWNER(S), CESSION; FORMER OWNER NAME: NOVARTIS VACCINES AND DIAGNOSTICS S.R.L.

Effective date: 20190205

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: PL

Payment date: 20190927

Year of fee payment: 15

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20200930

Year of fee payment: 16

Ref country code: IE

Payment date: 20200925

Year of fee payment: 16

Ref country code: NL

Payment date: 20200918

Year of fee payment: 16

Ref country code: PT

Payment date: 20200928

Year of fee payment: 16

Ref country code: FR

Payment date: 20200923

Year of fee payment: 16

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: BE

Payment date: 20200922

Year of fee payment: 16

Ref country code: CH

Payment date: 20200918

Year of fee payment: 16

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: TR

Payment date: 20201015

Year of fee payment: 16

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: AT

Payment date: 20200925

Year of fee payment: 16

Ref country code: ES

Payment date: 20201102

Year of fee payment: 16

Ref country code: IT

Payment date: 20201015

Year of fee payment: 16

Ref country code: DE

Payment date: 20200916

Year of fee payment: 16

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 602005044043

Country of ref document: DE

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: NL

Ref legal event code: MM

Effective date: 20211101

REG Reference to a national code

Ref country code: AT

Ref legal event code: MM01

Ref document number: 674189

Country of ref document: AT

Kind code of ref document: T

Effective date: 20211028

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20211031

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20211028

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20211028

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20220428

Ref country code: NL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20211101

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20211028

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20220503

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20211031

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20211031

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20211031

Ref country code: AT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20211028

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20211031

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20211028

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20211028

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20201028

REG Reference to a national code

Ref country code: ES

Ref legal event code: FD2A

Effective date: 20230207

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20211029